SP35 antibodies and uses thereof

ABSTRACT

Endogenous Sp35 is a negative regulator for neuronal survival, axon regeneration, oligodendrocyte differentiation and myelination (Negative Regulator). Molecules that block endogenous Sp35 function, such anti-Sp35 antibodies can be used as therapeutics for the treatment of neuron and oligodendrocyte dysfunction. The present invention provides antibodies specific for Sp35, and methods of using such antibodies as antagonists of endogenous Sp35 function. The invention further provides specific hybridoma and phage library-derived monoclonal antibodies, nucleic acids encoding these antibodies, and vectors and host cells comprising these antibodies. The invention further provides methods of promoting oligodendrocyte survival and myelination in a vertebrate, comprising administering to a vertebrate in need of such treatment an effective amount of an anti-Sp35 antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of International ApplicationNo. PCT/US2006/026271, filed Jul. 7, 2006, which claims the benefit ofU.S. Provisional Application No. 60/814,522, filed Jun. 19, 2006, U.S.Provisional Application No. 60/771,900, filed Feb. 10, 2006 and U.S.Provisional Application No. 60/697,336, filed Jul. 8, 2005, all of whichare incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to neurology, neurobiology and molecular biology.More particularly, this invention relates to molecules and methods fortreatment of neurological diseases, disorders and injuries such asspinal cord injury.

2. Background of the Invention

Axons and dendrites extend from neurons. The distal tip of an extendingaxon or neurite includes a specialized region, known as the growth cone.Growth cones sense the local environment and guide axonal growth towarda neuron's target cell. Growth cones respond to environmental cues, forexample, surface adhesiveness, growth factors, neurotransmitters andelectric fields. The growth cones generally advance at a rate of one totwo millimeters per day. The growth cone explores the area ahead of itand on either side, by means of elongations classified as lamellipodiaand filopodia. When an elongation contacts an unfavorable surface, itwithdraws. When an elongation contacts a favorable growth surface, itcontinues to extend and guides the growth cone in that direction. Whenthe growth cone reaches an appropriate target cell a synaptic connectionis created.

Nerve cell function is influenced by contact between neurons and othercells in their immediate environment (Rutishauser, et al., 1988,Physiol. Rev. 68:819). These cells include specialized glial cells,oligodendrocytes in the central nervous system (CNS), and Schwann cellsin the peripheral nervous system (PNS), which sheathe the neuronal axonwith myelin (Lemke, 1992, in An Introduction to Molecular Neurobiology,Z. Hall, Ed., p. 281, Sinauer).

CNS neurons have the inherent potential to regenerate after injury, butthey are inhibited from doing so by inhibitory proteins present inmyelin (Brittis et al., 2001, Neuron 30:11-14; Jones et al., 2002, J.Neurosci. 22:2792-2803; Grimpe et al., 2002, J. Neurosci.:22:3144-3160).

Several myelin inhibitory proteins found on oligodendrocytes have beencharacterized. Known examples of myelin inhibitory proteins includeNogoA (Chen et al., Nature, 2000, 403, 434-439; Grandpre et al., Nature2000, 403, 439-444), myelin associated glycoprotein (MAG) (McKerracheret al., 1994, Neuron 13:805-811; Mukhopadhyay et al., 1994, Neuron13:757-767) and oligodendrocyte glycoprotein (OM-gp), Mikol et al.,1988, J. Cell. Biol. 106:1273-1279). Each of these proteins has beenseparately shown to be a ligand for the neuronal Nogo receptor-1 (NgR1(Wang et al., Nature 2002, 417, 941-944; Grandpre et al., Nature 2000,403, 439-444; Chen et al., Nature, 2000, 403, 434-439; Domeniconi etal., Neuron 2002, published online Jun. 28, 2002).

Nogo receptor-1 (NgR1) is a GPI-anchored membrane protein that contains8 leucine rich repeats (Fournier et al., 2001, Nature 409:341-346). Uponinteraction with inhibitory proteins (e.g., NogoA, MAG and OM-gp), theNgR1 complex transduces signals that lead to growth cone collapse andinhibition of neurite outgrowth.

There is an unmet need for molecules and methods for inhibitingNgR1-mediated growth cone collapse and the resulting inhibition ofneurite outgrowth. Additionally there is a need for molecules whichincrease neuronal survival and axon regeneration. Particularly for thetreatment of disease, disorders or injuries which involve axonal injury,neuronal or oligodendrocyte cell death, demyelination or dymyelinationor generally relate to the nervous system.

Such diseases, disorders or injuries include, but are not limited to,multiple sclerosis (MS), progressive multifocal leukoencephalopathy(PML), encephalomyelitis (EPL), central pontine myelolysis (CPM),adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease(PMZ), Globoid cell Leucodystrophy (Krabbe's disease) and WallerianDegeneration, optic neuritis, transverse myelitis, amylotrophic lateralsclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson'sdisease, spinal cord injury, traumatic brain injury, post radiationinjury, neurologic complications of chemotherapy, stroke, acute ischemicoptic neuropathy, vitamin E deficiency, isolated vitamin E deficiencysyndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome,metachromatic leukodystrophy, trigeminal neuralgia, and Bell's palsy.Among these diseases, MS is the most widespread, affecting approximately2.5 million people worldwide.

MS generally begins with a relapsing-remitting pattern of neurologicinvolvement, which then progresses to a chronic phase with increasingneurological damage. MS is associated with the destruction of myelin,oligodendrocytes and axons localized to chronic lesions. Thedemyelination observed in MS is not always permanent and remyelinationhas been documented in early stages of the disease. Remyelination ofneurons requires oligodendrocytes.

Various disease-modifying treatments are available for MS, including theuse of corticosteroids and immunomodulators such as interferon beta andTysabri®. In addition, because of the central role of oligodendrocytesand myelination in MS, there have been efforts to develop therapies toincrease oligodendrocyte numbers or enhance myelination. See, e.g.,Cohen et al., U.S. Pat. No. 5,574,009; Chang et al., N. Engl. J. Med.346: 165-73 (2002). However, there remains an urgent need to deviseadditional therapies for MS and other demyelination and dismyelinationdisorders.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on the discovery that Sp35 (Sp35 is alsodesignated in the literature as LINGO-1 and LRRN6) is expressed inoligodendrocytes and neuronal cells and negatively regulatesoligodendrocyte/neuronal differentiation, survival and axon myelination.Furthermore, certain antagonists of Sp35 promote survival, proliferationand differentiation of oligodendrocytes and neuronal cells, as well asmyelination of neurons. Based on these discoveries, the inventionrelates generally to antibodies, antigen binding fragment or derivativesthereof which can be used as an antagonist of Sp35. Additionally, theinvention generally relates to methods for treating various disease,disorders or injuries associated with demyelination, dysmyelination,oligodendrocyte/neuronal cell death or axonal injury by theadministration of an Sp35 antagonist antibody or antigen bindingfragment.

In certain embodiments, the invention includes an isolated antibody orantigen binding fragment thereof which specifically binds to the sameSp35 epitope as a reference monoclonal antibody selected from the groupconsisting of 201′, 3A3, 3A6, 1A7, 1G7, 2B10, 2C11, 2F3, 3P1D10.2C3,3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2,3P4C5.1D8, 3P4C8.2G9, 30-C12 (Li01), 38-D01 (Li02), 35-E04 (Li03),36-C09 (Li04), 30-A11 (Li05), 34-F02 (Li06), 29-E07 (Li07), 34-G04(Li08), 36-A12 (Li09), 28-D02 (Li10), 30-B01 (Li11), 34-B03 (Li12),Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495 (L1a.2), 3563 (L1a.3), 3564(L1a.4), 3565 (L1a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 (L1a.8), 3569(L1a.9), 3570 (L1a.10), 3571 (L1a.11), 3582 (L1a.12), and 1968 (L1a.13).

Certain embodiments of the invention include an isolated polypeptidecomprising an immunoglobulin heavy chain variable region (VH) whereinthe CDR1, CDR2 and CDR3 regions are selected from the polypeptidesequences shown in Table 4 or at least 80%, 85%, 90 or 95% identical tothe polypeptide sequences shown in Table 4.

Certain embodiments of the invention include an isolated polypeptidecomprising an immunoglobulin light chain variable region (VL) whereinthe CDR1, CDR2 and CDR3 regions are selected from the polypeptidesequences shown in Table 5 or at least 80%, 85%, 90% or 95% identical tothe polypeptide sequences shown in Table 5.

Certain embodiments of the invention include an isolated polypeptidecomprising an immunoglobulin heavy chain variable region (VH) selectedfrom the group consisting of SEQ ID NOs: 158 to 172, 372, 376, 380 and384, as shown in Table 6, or at least 80%, 85%, 90% or 95% identical tosaid SEQ ID NOs: 158 to 172, 372, 376, 380 and 384 as shown in Table 6.

Certain embodiments of the invention include an isolated polypeptidecomprising an immunoglobulin light chain variable region (VL) selectedfrom the group consisting of SEQ ID NOs: 273 to 286, 373, 377, 381 and385, as shown in Table 8, or at least 80%, 85%, 90% or 95% identical tosaid SEQ ID NOs: 273 to 286, 373, 377, 381 and 385, as shown in Table 8.

In additional embodiments, the invention includes an isolatedpolynucleotide comprising a nucleic acid encoding an immunoglobulinheavy chain variable region (VH) wherein the CDR1, CDR2 and CDR3 regionsare selected from the group selected from the polynucleotide sequencesshown in Table 4 or at least 80%, 85%, 90 or 95% identical to thepolynucleotide sequences shown in Table 4.

In other embodiments, the invention includes an isolated polynucleotidecomprising a nucleic acid encoding an immunoglobulin light chainvariable region (VL) wherein the CDR1, CDR2 and CDR3 regions areselected from the polynucleotide sequences shown in Table 5 or at least80%, 85%, 90% or 95% identical to the polynucleotide sequences shown inTable 5.

Other embodiments of the invention include, an isolated polynucleotidecomprising a nucleic acid encoding an immunoglobulin heavy chainvariable region (VH) selected from the group consisting of SEQ ID NOs:173 to 184, 370, 374, 378 and 382, as shown in Table 7, or at least 80%,85%, 90% or 95% identical to said SEQ ID NOs: 173 to 184, 370, 374, 378and 382, as shown in Table 7.

Other embodiments of the invention include, an isolated polynucleotidecomprising a nucleic acid encoding an immunoglobulin light chainvariable region (VL) selected from the group consisting of SEQ ID NOs:185 to 194, 371, 375, 379 and 383, as shown in Table 9, or at least 80%,85%, 90% or 95% identical to said SEQ ID NOs: 185 to 194, 371, 375, 379and 383, as shown in Table 9.

In certain embodiments, the invention includes compositions comprisingthe antibodies or antigen binding fragments described herein.

In additional embodiments, the invention includes methods for treatingCNS injury, ALS, Huntington's disease, Alzheimer's disease, Parkinson'sdisease, diabetic neuropathy and stroke comprising administering to ananimal in need of said treatment an effective amount of an agentselected from the group consisting of an isolated Sp35 antibody orfragment thereof or compositions comprising said antibody or fragmentthereof.

In other embodiments, the invention includes methods for treatingdisease or disorders associated with inhibition of oligodendrocytegrowth or differentiation; demyelination or dysmyelination of CNSneurons including multiple sclerosis (MS), progressive multifocalleukoencephalopathy (PML), encephalomyelitis (EPL), central pontinemyelolysis (CPM), Wallerian Degeneration, adrenoleukodystrophy,Alexander's disease, and Pelizaeus Merzbacher disease (PMZ) byadministering to an animal in need of said treatment an effective amountof an agent selected from the group consisting of an isolated Sp35antibody or fragment thereof or compositions comprising said antibody orfragment thereof.

Other embodiments of the present invention include a method ofinhibiting signal transduction by Nogo receptor 1 (NgR1), comprisingcontacting the NgR1 with an effective amount of an agent selected fromthe group consisting of the isolated Sp35 antibody or fragment thereofor compositions comprising said antibody or fragment thereof.

Additional embodiments of the present invention include a method ofdecreasing inhibition of axonal growth of a central nervous system (CNS)neuron, comprising contacting the neuron with an effective amount of anagent selected from the group consisting of the isolated Sp35 antibodyor fragment thereof of or compositions comprising said antibody orfragment thereof.

Other embodiments of the present invention include a method ofinhibiting growth cone collapse of a CNS neuron, comprising contactingthe neuron with an effective amount of an agent selected from the groupconsisting of the isolated Sp35 antibody or fragment thereof orcompositions comprising said antibody or fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: SDS-PAGE gel showing immunoprecipitation of Sp35 by monoclonalantibodies 1A7 and 2F3.

FIG. 2: FACS result showing that MAbs 1A7 and 2F3 bound to COS-7 or 293cells expressing Sp35, but not to control cells with no Sp35 expression.

FIG. 3: MAbs 1A7 and 2F3 protected DRG neurons from myelin-mediatedinhibition of neurite outgrowth.

FIG. 4A-G: Immunohistochemical staining (“IHC”) of cocultures of DRGneurons and oligodendrocytes treated with monoclonal antibodies 1A7 and2F3, or control antibody. Panels D and E are enlargements of panels Band C, respectively. Staining with anti-βIII-tubulin antibody toidentify axons, or anti-MBP antibody to identify oligodendrocytes. F:Quantitation of MBP+ myelinating cells upon treatment of cocultures with1A7 or 2F3. G: Western blot analysis to quantify the MBP produced fromcocultures of DRG neurons and oligodendrocytes treated with monoclonalantibodies 1A7 and 2F3.

FIG. 5A-C: A: CC1 antibody staining of mouse oligodendrocytes incuprizone model. B. Anti-MBP protein antibody or luxol fast bluestaining of mouse neurons in cuprizone model. C: Quantitation of CC1antibody-positive oligodendrocytes at four weeks and 6 weeks.

FIG. 6: Surviving RGCs. Treatment with monoclonal antibody 1A7 Anti-Sp35antibody 1A7 treated animals showed significant neuronal survival (80%)when compared to control-antibody or PBS treated animals, which eachonly showed approximately 50% neuronal survival.

FIG. 7. BBB scores of mice receiving anti-Sp35 antibody 1A7 after spinalcord injury as described in Example 8.

FIG. 8. Western blot of co-cultured oligodendrocytes and DRGs afterincubation with anti-Sp35 antibodies Li05, Li06 and 3, 10 and 30 mg ofSp35-Fc (LINGO-1-Ig) as described in Example 9.

FIG. 9. Photographs of the optic nerves of A) Normal Rats; B) MyelinOligodendrocyte Glycoprotein (MOG) induced Experimental AutoimmuneEncephalomyelitis (EAE) rats; and C) Myelin Oligodendrocyte Glycoprotein(MOG) induced Experimental Autoimmune Encephalomyelitis (EAE) ratstreated with the Sp35 antibody 1A7. Electron micrographs of each opticnerve are shown below each photograph of the optic nerve.

FIG. 10. Graph of the number of regenerative neuronal fibers per sectioncounted in animals receiving an intravitreal injection of the Sp35antibody 1A7 after optic nerve crush.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an Sp35 antibody,” is understood torepresent one or more Sp35 antibodies. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” when referring to Sp35 antibodies or antibody polypeptides ofthe present invention include any polypeptides which retain at leastsome of the antigen-binding properties of the corresponding nativeantibody or polypeptide. Fragments of polypeptides of the presentinvention include proteolytic fragments, as well as deletion fragments,in addition to specific antibody fragments discussed elsewhere herein.Variants of Sp35 antibodies and antibody polypeptides of the presentinvention include fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally or benon-naturally occurring Non-naturally occurring variants may be producedusing art-known mutagenesis techniques. Variant polypeptides maycomprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of Sp35 antibodies and antibodypolypeptides of the present invention, are polypeptides which have beenaltered so as to exhibit additional features not found on the nativepolypeptide. Examples include fusion proteins. Variant polypeptides mayalso be referred to herein as “polypeptide analogs.” As used herein a“derivative” of an Sp35 antibody or antibody polypeptide refers to asubject polypeptide having one or more residues chemically derivatizedby reaction of a functional side group. Also included as “derivatives”are those peptides which contain one or more naturally occurring aminoacid derivatives of the twenty standard amino acids. For example,4-hydroxyproline may be substituted for proline; 5-hydroxylysine may besubstituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and ornithine maybe substituted for lysine.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refer to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan Sp35 antibody contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding anSp35 antibody or fragment, variant, or derivative thereof. Heterologouscoding regions include without limitation specialized elements ormotifs, such as a secretory signal peptide or a heterologous functionaldomain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

The present invention is directed to certain Sp35 antibodies, orantigen-binding fragments, variants, or derivatives thereof. Unlessspecifically referring to full-sized antibodies such asnaturally-occurring antibodies, the term “Sp35 antibodies” encompassesfull-sized antibodies as well as antigen-binding fragments, variants,analogs, or derivatives of such antibodies, e.g., naturally occurringantibody or immunoglobulin molecules or engineered antibody molecules orfragments that bind antigen in a manner similar to antibody molecules.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin comprises at least the variabledomain of a heavy chain, and normally comprises at least the variabledomains of a heavy chain and a light chain. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernable to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention. Allimmunoglobulin classes are clearly within the scope of the presentinvention, the following discussion will generally be directed to theIgG class of immunoglobulin molecules. With regard to IgG, a standardimmunoglobulin molecule comprises two identical light chain polypeptidesof molecular weight approximately 23,000 Daltons, and two identicalheavy chain polypeptides of molecular weight 53,000-70,000. The fourchains are typically joined by disulfide bonds in a “Y” configurationwherein the light chains bracket the heavy chains starting at the mouthof the “Y” and continuing through the variable region.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (V_(L)) and heavy (V_(H)) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (C_(L)) and the heavy chain (C_(H)1, C_(H)2or C_(H)3) confer important biological properties such as secretion,transplacental mobility, Fc receptor binding, complement binding, andthe like. By convention the numbering of the constant region domainsincreases as they become more distal from the antigen binding site oramino-terminus of the antibody. The N-terminal portion is a variableregion and at the C-terminal portion is a constant region; the C_(H)3and C_(L) domains actually comprise the carboxy-terminus of the heavyand light chain, respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the V_(L) domain and V_(H) domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen binding site. Thisquaternary antibody structure forms the antigen binding site present atthe end of each arm of the Y. More specifically, the antigen bindingsite is defined by three CDRs on each of the V_(H) and V_(L) chains. Insome instances, e.g., certain immunoglobulin molecules derived fromcamelid species or engineered based on camelid immunoglobulins, acomplete immunoglobulin molecule may consist of heavy chains only, withno light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448(1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops which connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable regionby one of ordinary skill in the art, since they have been preciselydefined (see, “Sequences of Proteins of Immunological Interest,” Kabat,E., et al., U.S. Department of Health and Human Services, (1983); andChothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which areincorporated herein by reference in their entireties).

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196:901-917 (1987), which are incorporated hereinby reference, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table 1 as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia V_(H) CDR1 31-35 26-32 V_(H) CDR250-65 52-58 V_(H) CDR3  95-102  95-102 V_(L) CDR1 24-34 26-32 V_(L) CDR250-56 50-52 V_(L) CDR3 89-97 91-96 ¹Numbering of all CDR definitions inTable 1 is according to the numbering conventions set forth by Kabat etal. (see below).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an Sp35 antibody or antigen-bindingfragment, variant, or derivative thereof of the present invention areaccording to the Kabat numbering system.

In camelid species, the heavy chain variable region, referred to asV_(H)H, forms the entire antigen-binding domain. The main differencesbetween camelid V_(H)H variable regions and those derived fromconventional antibodies (V_(H)) include (a) more hydrophobic amino acidsin the light chain contact surface of V_(H) as compared to thecorresponding region in V_(H)H, (b) a longer CDR3 in V_(H)H, and (c) thefrequent occurrence of a disulfide bond between CDR1 and CDR3 in V_(H)H.

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized, primatized, or chimericantibodies, single chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv), fragments comprising either aV_(L) or V_(H) domain, fragments produced by a Fab expression library,and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to Sp35 antibodies disclosed herein). ScFv molecules areknown in the art and are described, e.g., in U.S. Pat. No. 5,892,019.Immunoglobulin or antibody molecules of the invention can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, C_(H)1, C_(H)2, and C_(H)3domains. Also included in the invention are antigen-binding fragmentsalso comprising any combination of variable region(s) with a hingeregion, C_(H)1, C_(H)2, and C_(H)3 domains. Antibodies or immunospecificfragments thereof for use in the diagnostic and therapeutic methodsdisclosed herein may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, rabbit,goat, guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region may be condricthoid in origin (e.g.,from sharks). As used herein, “human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a C_(H)1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a C_(H)2 domain, a C_(H)3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a C_(H)1 domain; a polypeptide chaincomprising a C_(H)1 domain, at least a portion of a hinge domain, and aC_(H)2 domain; a polypeptide chain comprising a C_(H)1 domain and aC_(H)3 domain; a polypeptide chain comprising a C_(H)1 domain, at leasta portion of a hinge domain, and a C_(H)3 domain, or a polypeptide chaincomprising a C_(H)1 domain, at least a portion of a hinge domain, aC_(H)2 domain, and a C_(H)3 domain. In another embodiment, a polypeptideof the invention comprises a polypeptide chain comprising a C_(H)3domain. Further, a binding polypeptide for use in the invention may lackat least a portion of a C_(H)2 domain (e.g., all or part of a C_(H)2domain). As set forth above, it will be understood by one of ordinaryskill in the art that these domains (e.g., the heavy chain portions) maybe modified such that they vary in amino acid sequence from thenaturally occurring immunoglobulin molecule.

In certain Sp35 antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a C_(H)1 domain derived from an IgG1molecule and a hinge region derived from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a hinge region derived, inpart, from an IgG1 molecule and, in part, from an IgG3 molecule. Inanother example, a heavy chain portion can comprise a chimeric hingederived, in part, from an IgG1 molecule and, in part, from an IgG4molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. Preferably, thelight chain portion comprises at least one of a V_(L) or C_(L) domain.

Sp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof disclosed herein may be described or specified in terms of theepitope(s) or portion(s) of an antigen, e.g., a target polypeptide(Sp35) that they recognize or specifically bind. The portion of a targetpolypeptide which specifically interacts with the antigen binding domainof an antibody is an “epitope,” or an “antigenic determinant.” A targetpolypeptide may comprise a single epitope, but typically comprises atleast two epitopes, and can include any number of epitopes, depending onthe size, conformation, and type of antigen. Furthermore, it should benoted that an “epitope” on a target polypeptide may be or includenon-polypeptide elements, e.g., an “epitope may include a carbohydrateside chain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. In the present invention, peptide or polypeptide epitoperecognized by Sp35 antibodies of the present invention contains asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, or between about 15 to about 30 contiguous ornon-contiguous amino acids of Sp35.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody which“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

By way of non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds said first epitope with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second epitope. In another non-limiting example, an antibody may beconsidered to bind a first antigen preferentially if it binds the firstepitope with an affinity that is at least one order of magnitude lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstepitope preferentially if it binds the first epitope with an affinitythat is at least two orders of magnitude less than the antibody's K_(D)for the second epitope.

In another non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with an offrate (k(off)) that is less than the antibody's k(off) for the secondepitope. In another non-limiting example, an antibody may be consideredto bind a first epitope preferentially if it binds the first epitopewith an affinity that is at least one order of magnitude less than theantibody's k(off) for the second epitope. In another non-limitingexample, an antibody may be considered to bind a first epitopepreferentially if it binds the first epitope with an affinity that is atleast two orders of magnitude less than the antibody's k(off) for thesecond epitope.

An antibody or antigen-binding fragment, variant, or derivativedisclosed herein may be said to bind a target polypeptide disclosedherein or a fragment or variant thereof with an off rate (k(off)) ofless than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³sec⁻¹. More preferably, an antibody of the invention may be said to binda target polypeptide disclosed herein or a fragment or variant thereofwith an off rate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷sec⁻¹ or 10⁻⁷ sec⁻¹.

An antibody or antigen-binding fragment, variant, or derivativedisclosed herein may be said to bind a target polypeptide disclosedherein or a fragment or variant thereof with an on rate (k(on)) ofgreater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec¹ or5×10⁴ M⁻¹ sec⁻¹. More preferably, an antibody of the invention may besaid to bind a target polypeptide disclosed herein or a fragment orvariant thereof with an on rate (k(on)) greater than or equal to 10⁵ M⁻¹sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹ 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹sec⁻¹.

An antibody is said to competitively inhibit binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of animmunoglobulin molecule. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988)at pages 27-28. As used herein, the term “avidity” refers to the overallstability of the complex between a population of immunoglobulins and anantigen, that is, the functional combining strength of an immunoglobulinmixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual immunoglobulin molecules inthe population with specific epitopes, and also the valencies of theimmunoglobulins and the antigen. For example, the interaction between abivalent monoclonal antibody and an antigen with a highly repeatingepitope structure, such as a polymer, would be one of high avidity.

Sp35 antibodies or antigen-binding fragments, variants or derivativesthereof of the invention may also be described or specified in terms oftheir cross-reactivity. As used herein, the term “cross-reactivity”refers to the ability of an antibody, specific for one antigen, to reactwith a second antigen; a measure of relatedness between two differentantigenic substances. Thus, an antibody is cross reactive if it binds toan epitope other than the one that induced its formation. The crossreactive epitope generally contains many of the same complementarystructural features as the inducing epitope, and in some cases, mayactually fit better than the original.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Sp35 antibodies or antigen-binding fragments, variants or derivativesthereof of the invention may also be described or specified in terms oftheir binding affinity to a polypeptide of the invention. Preferredbinding affinities include those with a dissociation constant or Kd lessthan 5×10⁻² M, 10⁻²M, 5×10⁻³ M, 10⁻³M, 5×10⁻⁴M, 10⁻⁴M, 5×10⁻⁵M, 10⁻⁵ M,5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³ M,5×10⁻¹⁴M, 10⁻¹⁴ M, 5×10⁻¹⁵M, or 10⁻¹⁵M.

Sp35 antibodies or antigen-binding fragments, variants or derivativesthereof of the invention may be “multispecific,” e.g., bispecific,trispecific or of greater multispecificity, meaning that it recognizesand binds to two or more different epitopes present on one or moredifferent antigens (e.g., proteins) at the same time. Thus, whether anSp35 antibody is “monospecfic” or “multispecific,” e.g., “bispecific,”refers to the number of different epitopes with which a bindingpolypeptide reacts. Multispecific antibodies may be specific fordifferent epitopes of a target polypeptide described herein or may bespecific for a target polypeptide as well as for a heterologous epitope,such as a heterologous polypeptide or solid support material.

As used herein the term “valency” refers to the number of potentialbinding domains, e.g., antigen binding domains, present in an Sp35antibody, binding polypeptide or antibody. Each binding domainspecifically binds one epitope. When an Sp35 antibody, bindingpolypeptide or antibody comprises more than one binding domain, eachbinding domain may specifically bind the same epitope, for an antibodywith two binding domains, termed “bivalent monospecific,” or todifferent epitopes, for an antibody with two binding domains, termed“bivalent bispecific.” An antibody may also be bispecific and bivalentfor each specificity (termed “bispecific tetravalent antibodies”). Inanother embodiment, tetravalent minibodies or domain deleted antibodiescan be made.

Bispecific bivalent antibodies, and methods of making them, aredescribed, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, thedisclosures of all of which are incorporated by reference herein.Bispecific tetravalent antibodies, and methods of making them aredescribed, for instance, in WO 02/096948 and WO 00/44788, thedisclosures of both of which are incorporated by reference herein. Seegenerally, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos.4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al.,J. Immunol. 148:1547-1553 (1992).

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “V_(H) domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “C_(H)1 domain” includes the first (most amino terminal)constant region domain of an immunoglobulin heavy chain. The C_(H)1domain is adjacent to the V_(H) domain and is amino terminal to thehinge region of an immunoglobulin heavy chain molecule.

As used herein the term “C_(H)2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al. op. cit. The C_(H)2 domain is unique in that it isnot closely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two C_(H)2 domains of anintact native IgG molecule. It is also well documented that the C_(H)3domain extends from the C_(H)2 domain to the C-terminal of the IgGmolecule and comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the C_(H)1 domain to the C_(H)2 domain. Thishinge region comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al., J.Immunol. 161:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the C_(H)1 and C_(L)regions are linked by a disulfide bond and the two heavy chains arelinked by two disulfide bonds at positions corresponding to 239 and 242using the Kabat numbering system (position 226 or 229, EU numberingsystem).

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant invention) isobtained from a second species. In preferred embodiments the targetbinding region or site will be from a non-human source (e.g. mouse orprimate) and the constant region is human.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy and light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs may bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class andpreferably from an antibody from a different species. An engineeredantibody in which one or more “donor” CDRs from a non-human antibody ofknown specificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable region to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site. Given the explanations set forth in, e.g., U.S. Pat. Nos.5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well withinthe competence of those skilled in the art, either by carrying outroutine experimentation or by trial and error testing to obtain afunctional engineered or humanized antibody.

As used herein the term “properly folded polypeptide” includespolypeptides (e.g., Sp35 antibodies) in which all of the functionaldomains comprising the polypeptide are distinctly active. As usedherein, the term “improperly folded polypeptide” includes polypeptidesin which at least one of the functional domains of the polypeptide isnot active. In one embodiment, a properly folded polypeptide comprisespolypeptide chains linked by at least one disulfide bond and,conversely, an improperly folded polypeptide comprises polypeptidechains not linked by at least one disulfide bond.

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

As used herein, the terms “linked,” “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two ore more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature.) Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of such mRNA intopolypeptide(s). If the final desired product is a biochemical,expression includes the creation of that biochemical and any precursors.Expression of a gene produces a “gene product.” As used herein, a geneproduct can be either a nucleic acid, e.g., a messenger RNA produced bytranscription of a gene, or a polypeptide which is translated from atranscript. Gene products described herein further include nucleic acidswith post transcriptional modifications, e.g., polyadenylation, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, proteolytic cleavage, and the like.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of multiplesclerosis. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, andso on.

As used herein, phrases such as “a subject that would benefit fromadministration of an Sp35 antibody” and “an animal in need of treatment”includes subjects, such as mammalian subjects, that would benefit fromadministration of an Sp35 antibody used, e.g., for detection of an Sp35polypeptide (e.g., for a diagnostic procedure) and/or from treatment,i.e., palliation or prevention of a disease such as MS, with an Sp35antibody. As described in more detail herein, the Sp35 antibody can beused in unconjugated form or can be conjugated, e.g., to a drug,prodrug, or an isotope.

II. Sp35

Naturally occurring human Sp35 (Sp35) is a glycosylated central nervoussystem-specific protein which is predicted to have 614 amino acids (SEQID NO: 2), including a 33 amino acid signal sequence. Sp 35 is alsoknown in the art by the names LINGO-1, LRRN6, LRRN6A, FLJ14594, LERN1,MGC17422 and UNQ201. The human, full-length wild-type Sp35 polypeptidecontains an LRR domain consisting of 14 leucine-rich repeats (includingN- and C-terminal caps), an Ig domain, a transmembrane region, and acytoplasmic domain. The cytoplasmic domain contains a canonical tyrosinephosphorylation site. In addition, the naturally occurring Sp35 proteincontains a signal sequence, a short basic region between the LRRCT andIg domain, and a transmembrane region between the Ig domain and thecytoplasmic domain. The human Sp35 gene (SEQ ID NO:1) containsalternative translation start codons, so that six additional aminoacids, i.e., MQVSKR (SEQ ID NO: 3) may or may not be present at theN-terminus of the Sp35 signal sequence. Table 2 lists the Sp35 domainsand other regions, according to amino acid residue number, based on theSp35 amino acid sequence presented herein as SEQ ID NO: 2. The Sp35polypeptide is characterized in more detail in PCT Publication No. WO2004/085648, which is incorporated herein by reference in its entirety.

TABLE 2 Sp35 Domains Domain or Region Beginning Residue Ending ResidueSignal Sequence 1 33 or 35 LRRNT 34 or 36 64 LRR 66 89 LRR 90 113 LRR114 137 LRR 138 161 LRR 162 185 LRR 186 209 LRR 210 233 LRR 234 257 LRR258 281 LRR 282 305 LRR 306 329 LRR 330 353 LRRCT 363 414 or 416 Basic415 or 417 424 Ig 419 493 Connecting sequence 494 551 Transmembrane 552576 Cytoplasmic 577 614

Tissue distribution and developmental expression of Sp35 has beenstudied in humans and rats. Sp35 biology has been studied in anexperimental animal (rat) model. Expression of rat Sp35 is localized toneurons and oligodendrocytes, as determined by northern blot andimmuno-histochemical staining. Rat Sp35 mRNA expression level isregulated developmentally, peaking shortly after birth, i.e., ca.postnatal day one. In a rat spinal cord transection injury model, Sp35is up-regulated at the injury site, as determined by RT-PCR. See Mi etal. Nature Neurosci. 7:221-228 (2004).

In the context of the amino acids comprising the various structural andfunctional domains of an Sp35 polypeptide, the term “about” includes theparticularly recited value and values larger or smaller by several(e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acids. Since the locationof these domains as listed in Table 1 have been predicted by computergraphics, one of ordinary skill would appreciate that the amino acidresidues constituting the domains may vary slightly (e.g., by about 1 to15 residues) depending on the criteria used to define the domain.

The inventors have discovered that full-length, wild-type Sp35 binds toNgR1. See PCT Publication No. WO 2004/085648. The inventors have alsodiscovered that Sp35 is expressed in oligodendrocytes and that the Sp35protein is involved in the regulation of oligodendrocyte-mediatedmyelination of axons. See U.S Patent Publication No. 2006/0009388 A1,which is incorporated herein by reference in its entirety.

The nucleotide sequence for the full-length Sp35 molecule is as follows:

ATGCTGGCGGGGGGCGTGAGGAGCATGCCCAGCCCCCTCCTGGCCTGCTGGCAGCCCATCCTCCTGCTGG(SEQ ID NO: 1)TGCTGGGCTCAGTGCTGTCAGGCTCGGCCACGGGCTGCCCGCCCCGCTGCGAGTGCTCCGCCCAGGACCGCGCTGTGCTGTGCCACCGCAAGCGCTTTGTGGCAGTCCCCGAGGGCATCCCCACCGAGACGCGCCTGCTGGACCTAGGCAAGAACCGCATCAAAACGCTCAACCAGGACGAGTTCGCCAGCTTCCCGCACCTGGAGGAGCTGGAGCTCAACGAGAACATCGTGAGCGCCGTGGAGCCCGGCGCCTTCAACAACCTCTTCAACCTCCGGACGCTGGGTCTCCGCAGCAACCGCCTGAAGCTCATCCCGCTAGGCGTCTTCACTGGCCTCAGCAACCTGACCAAGCTGGACATCAGCGAGAACAAGATTGTTATCCTGCTGGACTACATGTTTCAGGACCTGTACAACCTCAAGTCACTGGAGGTTGGCGACAATGACCTCGTCTACATCTCTCACCGCGCCTTCAGCGGCCTCAACAGCCTGGAGCAGCTGACGCTGGAGAAATGCAACCTGACCTCCATCCCCACCGAGGCGCTGTCCCACCTGCACGGCCTCATCGTCCTGAGGCTCCGGCACCTCAACATCAATGCCATCCGGGACTACTCCTTCAAGAGGCTCTACCGACTCAAGGTCTTGGAGATCTCCCACTGGCCCTACTTGGACACCATGACACCCAACTGCCTCTACGGCCTCAACCTGACGTCCCTGTCCATCACACACTGCAATCTGACCGCTGTGCCCTACCTGGCCGTCCGCCACCTAGTCTATCTCCGCTTCCTCAACCTCTCCTACAACCCCATCAGCACCATTGAGGGCTCCATGTTGCATGAGCTGCTCCGGCTGCAGGAGATCCAGCTGGTGGGCGGGCAGCTGGCCGTGGTGGAGCCCTATGCCTTCCGCGGCCTCAACTACCTGCGCGTGCTCAATGTCTCTGGCAACCAGCTGACCACACTGGAGGAATCAGTCTTCCACTCGGTGGGCAACCTGGAGACACTCATCCTGGACTCCAACCCGCTGGCCTGCGACTGTCGGCTCCTGTGGGTGTTCCGGCGCCGCTGGCGGCTCAACTTCAACCGGCAGCAGCCCACGTGCGCCACGCCCGAGTTTGTCCAGGGCAAGGAGTTCAAGGACTTCCCTGATGTGCTACTGCCCAACTACTTCACCTGCCGCCGCGCCCGCATCCGGGACCGCAAGGCCCAGCAGGTGTTTGTGGACGAGGGCCACACGGTGCAGTTTGTGTGCCGGGCCGATGGCGACCCGCCGCCCGCCATCCTCTGGCTCTCACCCCGAAAGCACCTGGTCTCAGCCAAGAGCAATGGGCGGCTCACAGTCTTCCCTGATGGCACGCTGGAGGTGCGCTACGCCCAGGTACAGGACAACGGCACGTACCTGTGCATCGCGGCCAACGCGGGCGGCAACGACTCCATGCCCGCCCACCTGCATGTGCGCAGCTACTCGCCCGACTGGCCCCATCAGCCCAACAAGACCTTCGCTTTCATCTCCAACCAGCCGGGCGAGGGAGAGGCCAACAGCACCCGCGCCACTGTGCCTTTCCCCTTCGACATCAAGACCCTCATCATCGCCACCACCATGGGCTTCATCTCTTTCCTGGGCGTCGTCCTCTTCTGCCTGGTGCTGCTGTTTCTCTGGAGCCGGGGCAAGGGCAACACAAAGCACAACATCGAGATCGAGTATGTGCCCCGAAAGTCGGACGCAGGCATCAGCTCCGCCGACGCGCCCCGCAAGTTCAACATGAAGATGATATGA.

The polypeptide sequence for the full-length Sp35 polypeptide is asfollows:

MLAGGVRSMPSPLLACWQPILLLVLGSVLSGSATGCPPRCECSAQDRAVLCHRKRFVAVPEGIPTETRL(SEQ ID NO: 2)LDLGKNRIKTLNQDEFASFPHLEELELNENIVSAVEPGAFNNLFNLRTLGLRSNRLKLIPLGVFTGLSNLTKLDISENKIVILLDYMFQDLYNLKSLEVGDNDLVYISHAAFSGLNSLEQLTLEKCNLTSIPTEALSHLHGLIVLRLRHLNINAIRDYSFKRLYRLKVLEISHWPYLDTMTPNCLYGLNLTSLSITHCNLTAVPYLAVRHLVYLRFLNLSYNPISTIEGSMLHELLRLQEIQLVGGQLAVVEPYAFRGLNYLRVLNVSGNQLTTLEESVFHSVGNLETLILDSNPLACDCRLLWVFRRRWRLNFNRQQPTCATPEFVQGKEFAAFPDVLLPNYFTCRRARIRDRKAQQVFVDEGHTVQFVCRADGDPPPAILWLSPRKHLVSAKSNGRLTVFPDGTLEVRYAQVQDNGTYLCIAANAGGNDSMPAHLHVRSYSPDWPHQPNKTFAFISNQPGEGEANSTRATVPFPFDIKTLIIATTMGFISFLGVVLFCLVLLFLWSRGKGNTKHIEIEYVPRKSDAGISSADAPRKNMKMI.

III. Sp35 Antibodies

In one embodiment, the present invention is directed to Sp35 antibodies,or antigen-binding fragments, variants, or derivatives thereof. Forexample, the present invention includes at least the antigen-bindingdomains of certain monoclonal antibodies, and fragments, variants, andderivatives thereof shown in Tables 3A and 3B.

Table 3A describes the regions of the Sp35 polypeptide that are bound bycertain full-length phage library derived antibodies. These antibodieshave the same variable regions as the Fab fragments derived from PhageDisplay Library-1, as indicated in Table 3B (e.g. D05 in Table 3A hasthe same variable region as Li05 in Table 3B, D06 in Table 3A has thesame variable region as Li06 in Table 3B, etc.). The antibodies weretested for binding Sp35 fragments as defined in Table 3A, using methodswell known in the art.

Table 3B describes the ability of the named monoclonal antibodies or Fabfragments to detect Sp35 in various assays such as: FluorescentActivated Cell Sorting (FACS), Immunoprecipitation (IP), Western blotanalysis, Immunohistochemistry (IHC) and Enzyme Linked ImmunosorbentAssay (ELISA). Detailed protocols for performing these assays aredescribed herein or are well known and understood by those of ordinaryskill in the art. Hybridoma-derived monoclonal antibodies listed inTable 3B were produced by injection of soluble Sp35 into mice and thenisolated using hybridoma technology which is well known in the art anddescribed herein. Monoclonal antibodies and antibody Fab fragmentslisted in Table 3B were isolated from two different phage displaylibraries using techniques known in the art.

TABLE 3A D03 D05 D06 D08 D11 D13 D33 (Li03 (Li05 (Li06 (Li08 (Li03 (Li13(Li33 Sp35 Variable Variable Variable Variable Variable VariableVariable Fragment Region) Region) Region) Region) Region) Region)Region) 1-432 rat + + + − + − + Fc 417-493 − +/− +/− − − − − rat FcAP-Sp35 N/D + −/+ −/+ N/D N/D N/D (1-419) AP-Sp35 N/D − − − N/D N/D NID(418-498) 417-498 − − − − − − − human Fc 417-503 − − − − − − − human Fc363-498 − − − − − − − human Fc 244-498 − − − − − − − human Fc

TABLE 3B SP35 MONOCLONAL ANTIBODIES Western IHC on ELISA FACsImmunoprecipitation mouse/ Transfected Cells IHC on Tissues 34- 417-419- 1- 34- huSp35 mSp35 sp35Fc huSp35 mSp35 huSp35 rat sp35 huSp35mSp35 WT (parafin) KO (parafin) 417 493 495 532 532 HYBRIDOMA-DERIVEDMONOCLONAL ANTIBODIES 201′ yes yes No (mouse N/A N/A yes yes and rat)3A3 − − + − − no No (mouse no no yes yes and rat) 3A6 ++ +/− ++ +++ −/+no No (mouse yes w no and rat) background 1A7 ++ − ++ +++ −/+ no No(mouse yes w no +/− yes yes and rat) background 1G7 ++ +/− ++ +++ + noNo (mouse yes w no and rat) background 2B10 ++ +/− + +++ −/+ no No(mouse yes w no yes yes and rat) background 2C11 − − − − − no No (mouseno no and rat) 2F3 +/− +/− +++ +++ yes yes with yes yes yes yes yes yesover-expressed mSp35 3P1B1.1F9 +++ − 3P1D10.2C3 +++ − +/− yes yes3P1E11.3B7 +++ − +/− yes yes 3P2C6. 3G10.2H7 +++ − +/− yes yes 3P2C9.2G4+++ − +/− yes yes 3P4A6.1D9 +++ − +/− yes yes 3P4A1.2B9 +++ − 3P4C2.2D2+++ +++ yes yes 3P4C5.1D8 +++ − +/− yes yes 3P4C8.2G9 +++ +++ yes Yesyes yes (mouse) 7P1D5.1G9 + + +++ +++ no No (mouse) 1B6.4 +++ +++ ++++++ no No (upper (lower band) (mouse) band) 2C7.2 +++ +++ +++ +++ no No(upper (lower (mouse) band) band) 2D6.1 ++ ++ − − no No (binds to (bindsto (mouse) 293 cells) 293 cells) 2F7.3 ++ ++ +++ +++ yes Yes (lower(lower (mouse) band) band) 2H3.2 ++ ++ +++ +++ yes Yes (lower (lower(mouse) band) band) 3C11.1 ++ ++ +++ +++ yes Yes (lower (lower (mouse)band) band) 3E3.1 +++ +++ +++ +++ no No (upper (lower (mouse) band)band) 3H11.2 ++ ++ +++ +++ yes Yes (lower (lower (mouse) band) band)3G8.1 + + +++ +++ no No (upper (mouse) band) 2B8.1 ++ ++ +++ + no No(upper (lower (mouse) band) band) 3B5.2 +++ +++ +++ +++ no No (upper(mouse) band) PHAGE DISPLAY LIBRARY-1 DERIVED MONOCLONAL Fab FRAGMENTS30-C12 (Li01) ++ ++ 38-D01 (Li02) −/+ −/+ 35-E04 (Li03) ++ +++ 36-C09(Li04) −/+ −/+ 30-A11 (Li05) + ++ ++ ++ 34-F02 (Li06) ++ ++ 29-E07(Li07) ++ ++ 34-G04 (Li08) +/− + ++ ++ 36-A12 (Li09) − − 28-D02 (Li10)−/+ +/− 30-B01 (Li11) ++ ++ ++ 34-B03 (Li12) + + PHAGE DISPLAY LIBRARY-2DERIVED MONOCLONAL Fab FRAGMENTS 3383 (1) + − N/A N/A yes yes yes yes3495(2) + − yes no faint N/A yes yes +/− yes yes 3563 (3) + no no yesyes 3564 (4) + no no yes yes 3565 (5) + no no yes yes 3566 (6) + yesvery faint yes yes +/− yes yes 3567 (7) + yes no yes yes +/− yes yes3568 (8) + no no yes yes 3569 (9) + no no yes yes 3570 (10) + no no yesyes 3571 (11) + no no 3582 (12) + no no yes yes 1968 (13) +/− − ++ weakno very faint yes w bg yes yes +/− yes yes 3011 − +/− only stain veryfew cells faint 3012 − − no no 3013 sticky + yes w high bg yes 3418sticky yes w high bg yes 3422 − very faint yes w high bg 3562 sticky nono PHAGE DISPLAY LIBRARY-1 DERIVED COMPLETE MONOCLONAL ANTIBODIES D05 ++D07 +++ D08 ++ D10 +++ D11 +++ Key: huSp35 = human Sp35 protein mSp35 =mouse Sp35 protein WT = wild-type KO = knock-out IHC =immunohistochemistry FACS = Fluorescent Activated Cell Sorting

As used herein, the term “antigen binding domain” includes a site thatspecifically binds an epitope on an antigen (e.g., an epitope of Sp35).The antigen binding domain of an antibody typically includes at least aportion of an immunoglobulin heavy chain variable region and at least aportion of an immunoglobulin light chain variable region. The bindingsite formed by these variable regions determines the specificity of theantibody.

The present invention is more specifically directed to an Sp35 antibody,or antigen-binding fragment, variant or derivatives thereof, where theSp35 antibody binds to the same epitope as a monoclonal antibodyselected from the group consisting of 201′, 3A3, 3A6, 1A7, 1G7, 2B10,2C11, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9,3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (Li01), 38-D01(Li02), 35-E04 (Li03), 36-C09 (Li04), 30-A11 (Li05), 34-F02 (Li06),29-E07 (Li07), 34-G04 (Li08), 36-A 12 (Li09), 28-D02 (Li10), 30-B01(Li11), 34-B03 (Li12), Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495(L1a.2), 3563 (L1a.3), 3564 (L1a.4), 3565 (L1a.5), 3566 (L1a.6), 3567(L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570 (L1a.10), 3571 (L1a.11), 3582(L1a.12), and 1968 (L1a.13).

The invention is further drawn to an Sp35 antibody, or antigen-bindingfragment, variant or derivatives thereof, where the Sp35 antibodycompetitively inhibits a monoclonal antibody selected from the groupconsisting of 201′, 3A3, 3A6, 1A7, 1G7, 2B10, 2C11, 2F3, 3P1D10.2C3,3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2,3P4C5.1D8, 3P4C8.2G9, 30-C12 (L100), 38-D01 (Li02), 35-E04 (Li03),36-C09 (Li04), 30-A11 (Li05), 34-F02 (Li06), 29-E07 (Li07), 34-G04(Li08), 36-A12 (Li09), 28-D02 (Li10), 30-B01 (Li11), 34-B03 (Li12),Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495 (L1a.2), 3563 (L1a.3), 3564(L1a.4), 3565 (L1a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 (L1a.8), 3569(L1a.9), 3570 (L1a.10), 3571 (L1a.11), 3582 (L1a.12), and 1968 (L1a.13)from binding to Sp35.

The invention is also drawn to an Sp35 antibody, or antigen-bindingfragment, variant or derivatives thereof, where the Sp35 antibodycomprises at least the antigen binding region of a monoclonal antibodyselected from the group consisting of 201′, 3A3, 3A6, 1A7, 1G7, 2B10,2C11, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9,3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (L100), 38-D01(Li02), 35-E04 (Li03), 36-C09 (Li04), 30-A11 (Li05), 34-F02 (Li06),29-E07 (Li07), 34-G04 (Li08), 36-A12 (Li09), 28-D02 (Li10), 30-B01(Li11), 34-B03 (Li12), Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495(L1a.2), 3563 (L1a.3), 3564 (L1a.4), 3565 (L1a.5), 3566 (L1a.6), 3567(L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570 (L1a.10), 3571 (L1a.11), 3582(L1a.12), and 1968 (L1a.13).

In certain embodiments, the present invention is directed to anantibody, or antigen-binding fragment, variant, or derivative thereofwhich specifically or preferentially binds to a particular Sp35polypeptide fragment or domain. Such Sp35 polypeptide fragments include,but are not limited to, an Sp35 polypeptide comprising, consistingessentially of, or consisting of amino acids 34 to 532; 34 to 417; 34 to425; 34 to 493; 66 to 532; 66 to 417; 66 to 426; 66 to 493; 66 to 532;417 to 532; 417 to 425 (the Sp35 basic region); 417 to 493; 417 to 532;419 to 493 (the Sp351 g region); or 425 to 532 of SEQ ID NO:2; or anSp35 variant polypeptide at least 70%, 75%, 80%, 85%, 90%, or 95%identical to amino acids 34 to 532; 34 to 417; 34 to 425; 34 to 493; 66to 532; 66 to 417; 66 to 426; 66 to 493; 66 to 532; 417 to 532; 417 to425 (the Sp35 basic region); 417 to 493; 417 to 532; 419 to 493 (theSp351 g region); or 425 to 532 of SEQ ID NO:2.

Additional Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of one or moreleucine-rich-repeats (LRR) of Sp35. Such fragments, include, forexample, fragments comprising, consisting essentially of, or consistingof amino acids 66 to 89; 66 to 113; 66 to 137; 90 to 113; 114 to 137;138 to 161; 162 to 185; 186 to 209; 210 to 233; 234 to 257; 258 to 281;282 to 305; 306 to 329; or 330 to 353 of SEQ ID NO:2. Correspondingfragments of a variant Sp35 polypeptide at least 70%, 75%, 80%, 85%,90%, or 95% identical to amino acids 66 to 89; 66 to 113; 90 to 113; 114to 137; 138 to 161; 162 to 185; 186 to 209; 210 to 233; 234 to 257; 258to 281; 282 to 305; 306 to 329; or 330 to 353 of SEQ ID NO:2 are alsocontemplated.

Additional Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of one or morecysteine rich regions flanking the LRR of Sp35. Such fragments, include,for example, a fragment comprising, consisting essentially of, orconsisting of amino acids 34 to 64 of SEQ ID NO:2 (the N-terminal LRRflanking region (LRRNT)), or a fragment comprising, consistingessentially of, or consisting of amino acids 363 to 416 of SEQ ID NO:2(the C-terminal LRR flanking region (LRRCT)), amino acids Correspondingfragments of a variant Sp35 polypeptide at least 70%, 75%, 80%, 85%,90%, or 95% identical to amino acids 34 to 64 and 363 to 416 of SEQ IDNO:2 are also contemplated.

As known in the art, “sequence identity” between two polypeptides isdetermined by comparing the amino acid sequence of one polypeptide tothe sequence of a second polypeptide. When discussed herein, whether anyparticular polypeptide is at least about 70%, 75%, 80%, 85%, 90% or 95%identical to another polypeptide can be determined using methods andcomputer programs/software known in the art such as, but not limited to,the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). BESTFIT uses the local homology algorithmof Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981),to find the best segment of homology between two sequences. When usingBESTFIT or any other sequence alignment program to determine whether aparticular sequence is, for example, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference polypeptide sequence and that gaps in homologyof up to 5% of the total number of amino acids in the reference sequenceare allowed.

Additional Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 41to 525 of SEQ ID NO:2; 40 to 526 of SEQ ID NO:2; 39 to 527 of SEQ IDNO:2; 38 to 528 of SEQ ID NO:2; 37 to 529 of SEQ ID NO:2; 36 to 530 ofSEQ ID NO:2; 35 to 531 of SEQ ID NO:2; 34 to 531 of SEQ ID NO:2; 46 to520 of SEQ ID NO:2; 45 to 521 of SEQ ID NO:2; 44 to 522 of SEQ ID NO:2;43 to 523 of SEQ ID NO:2; and 42 to 524 of SEQ ID NO:2.

Still additional Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 1 to33 of SEQ ID NO:2; 1 to 35 of SEQ ID NO:2; 34 to 64 of SEQ ID NO:2; 36to 64 of SEQ ID NO:2; 66 to 89 of SEQ ID NO:2; 90 to 113 of SEQ ID NO:2;114 to 137 of SEQ ID NO:2; 138 to 161 of SEQ ID NO:2; 162 to 185 of SEQID NO:2; 186 to 209 of SEQ ID NO:2; 210 to 233 of SEQ ID NO:2; 234 to257 of SEQ ID NO:2; 258 to 281 of SEQ ID NO:2; 282 to 305 of SEQ IDNO:2; 306 to 329 of SEQ ID NO:2; 330 to 353 of SEQ ID NO:2; 363 to 416of SEQ ID NO:2; 417 to 424 of SEQ ID NO:2; 419 to 493 of SEQ ID NO:2;and 494 to 551 of SEQ ID NO:2.

Further still, Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 1 to33 of SEQ ID NO:2; 1 to 35 of SEQ ID NO:2; 1 to 64 of SEQ ID NO:2; 1 to89 of SEQ ID NO:2; 1 to 113 of SEQ ID NO:2; 1 to 137 of SEQ ID NO:2; 1to 161 of SEQ ID NO:2; 1 to 185 of SEQ ID NO:2; 1 to 209 of SEQ ID NO:2;1 to 233 of SEQ ID NO:2; 1 to 257 of SEQ ID NO:2; 1 to 281 of SEQ IDNO:2; 1 to 305 of SEQ ID NO:2; 1 to 329 of SEQ ID NO:2; 1 to 353 of SEQID NO:2; 1 to 416 of SEQ ID NO:2; 1 to 424 of SEQ ID NO:2; 1 to 493 ofSEQ ID NO:2; 1 to 551 of SEQ ID NO:2; 1 to 531 of SEQ ID NO:2 and 1 to532 of SEQ ID NO:2.

Additional Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 34to 64 of SEQ ID NO:2; 34 to 89 of SEQ ID NO:2; 34 to 113 of SEQ ID NO:2;34 to 137 of SEQ ID NO:2; 34 to 161 of SEQ ID NO:2; 34 to 185 of SEQ IDNO:2; 34 to 209 of SEQ ID NO:2; 34 to 233 of SEQ ID NO:2; 34 to 257 ofSEQ ID NO:2; 34 to 281 of SEQ ID NO:2; 34 to 305 of SEQ ID NO:2; 34 to329 of SEQ ID NO:2; 34 to 353 of SEQ ID NO:2; 34 to 416 of SEQ ID NO:2;34 to 424 of SEQ ID NO:2; 34 to 493 of SEQ ID NO:2; and 34 to 551 of SEQID NO:2.

More additional Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 34to 530 of SEQ ID NO:2; 34 to 531 of SEQ ID NO:2; 34 to 532 of SEQ IDNO:2; 34 to 533 of SEQ ID NO:2; 34 to 534 of SEQ ID NO:2; 34 to 535 ofSEQ ID NO:2; 34 to 536 of SEQ ID NO:2; 34 to 537 of SEQ ID NO:2; 34 to538 of SEQ ID NO:2; 34 to 539 of SEQ ID NO:2; 30 to 532 of SEQ ID NO:2;31 to 532 of SEQ ID NO:2; 32 to 532 of SEQ ID NO:2; 33 to 532 of SEQ IDNO:2; 34 to 532 of SEQ ID NO:2; 35 to 532 of SEQ ID NO:2; 36 to 532 ofSEQ ID NO:2; 30 to 531 of SEQ ID NO:2; 31 to 531 of SEQ ID NO:2; 32 to531 of SEQ ID NO:2; 33 to 531 of SEQ ID NO:2; 34 to 531 of SEQ ID NO:2;35 to 531 of SEQ ID NO:2; and 36 to 531 of SEQ ID NO:2.

Further still, Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 36to 64 of SEQ ID NO:2; 36 to 89 of SEQ ID NO:2; 36 to 113 of SEQ ID NO:2;36 to 137 of SEQ ID NO:2; 36 to 161 of SEQ ID NO:2; 36 to 185 of SEQ IDNO:2; 36 to 209 of SEQ ID NO:2; 36 to 233 of SEQ ID NO:2; 36 to 257 ofSEQ ID NO:2; 36 to 281 of SEQ ID NO:2; 36 to 305 of SEQ ID NO:2; 36 to329 of SEQ ID NO:2; 36 to 353 of SEQ ID NO:2; 36 to 416 of SEQ ID NO:2;36 to 424 of SEQ ID NO:2; 36 to 493 of SEQ ID NO:2; and 36 to 551 of SEQID NO:2.

Additional Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 36to 530 of SEQ ID NO:2; 36 to 531 of SEQ ID NO:2; 36 to 532 of SEQ IDNO:2; 36 to 533 of SEQ ID NO:2; 36 to 534 of SEQ ID NO:2; 36 to 535 ofSEQ ID NO:2; 36 to 536 of SEQ ID NO:2; 36 to 537 of SEQ ID NO:2; 36 to538 of SEQ ID NO:2; and 36 to 539 of SEQ ID NO:2.

More Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 417to 493 of SEQ ID NO:2; 417 to 494 of SEQ ID NO:2; 417 to 495 of SEQ IDNO:2; 417 to 496 of SEQ ID NO:2; 417 to 497 of SEQ ID NO:2; 417 to 498of SEQ ID NO:2; 417 to 499 of SEQ ID NO:2; 417 to 500 of SEQ ID NO:2;417 to 492 of SEQ ID NO:2; 417 to 491 of SEQ ID NO:2; 412 to 493 of SEQID NO:2; 413 to 493 of SEQ ID NO:2; 414 to 493 of SEQ ID NO:2; 415 to493 of SEQ ID NO:2; 416 to 493 of SEQ ID NO:2; 411 to 493 of SEQ IDNO:2; 410 to 493 of SEQ ID NO:2; 410 to 494 of SEQ ID NO:2; 411 to 494of SEQ ID NO:2; 412 to 494 of SEQ ID NO:2; 413 to 494 of SEQ ID NO:2;414 to 494 of SEQ ID NO:2; 415 to 494 of SEQ ID NO:2; 416 to 494 of SEQID NO:2; 417 to 494 of SEQ ID NO:2; and 418 to 494 of SEQ ID NO:2.

In an additional embodiment Sp35 peptide fragments to which certainantibodies, or antigen-binding fragments, variants, or derivativesthereof of the present invention bind include, an Sp35 polypeptidecomprising, consisting essentially of, or consisting of peptides of theIg domain of Sp35 or fragments, variants, or derivatives of suchpolypeptides. Specifically, polypeptides comprising, consistingessentially of, or consisting of the following polypeptide sequences:ITX₁X₂X₃ (SEQ ID NO:287), ACX₁X₂X₃ (SEQ ID NO:288), VCX₁X₂X₃ (SEQ IDNO:289) and SPX₁X₂X₃ (SEQ ID NO:290) where X₁ is lysine, arginine,histidine, glutamine, or asparagine, X₂ is lysine, arginine, histidine,glutamine, or asparagine and X₃ is lysine, arginine, histidine,glutamine, or asparagine. For example, Sp35 peptide fragments to whichcertain antibodies, or antigen-binding fragments, variants, orderivatives thereof of the present invention bind include, thosefragments comprising, consisting essentially of, or consisting of thefollowing polypeptide sequences: SPRKH (SEQ ID NO:291), SPRKK (SEQ IDNO:292), SPRKR (SEQ ID NO:293), SPKKH (SEQ ID NO:294), SPHKH (SEQ IDNO:295), SPRRH (SEQ ID NO:296), SPRHH (SEQ ID NO:297), SPRRR (SEQ IDNO:298), SPHHH (SEQ ID NO:299) SPKKK (SEQ ID NO:300), LSPRKH (SEQ IDNO:301), LSPRKK (SEQ ID NO:302), LSPRKR (SEQ ID NO:303), LSPKKH (SEQ IDNO:304), LSPHKH (SEQ ID NO:305), LSPRRH (SEQ ID NO:306), LSPRHH (SEQ IDNO:307), LSPRRR (SEQ ID NO:308), LSPHHH (SEQ ID NO:309) LSPKKK (SEQ IDNO:310), WLSPRKH (SEQ ID NO:311), WLSPRKK (SEQ ID NO:312), WLSPRKR (SEQID NO:313), WLSPKKH (SEQ ID NO:314), WLSPHKH (SEQ ID NO:315), WLSPRRH(SEQ ID NO:316), WLSPRHH (SEQ ID NO:317), WLSPRRR (SEQ ID NO:318),WLSPHHH (SEQ ID NO:319) WLSPKKK (SEQ ID NO:320). These Sp35 polypeptidesinclude the basic “RKH loop” (Arginine-Lysine-Histidine amino acids456-458) in the Ig domain of Sp35. Additional Sp35 peptides whichinclude a basic tripeptide are ITPKRR (SEQ ID NO:321), ACHHK (SEQ IDNO:322) and VCHHK (SEQ ID NO:323).

Additional Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, an Sp35 polypeptide comprising,consisting essentially of, or consisting of peptides of the Ig domain ofSp35 or fragments, variants, or derivatives of such polypeptides.Specifically, peptides comprising, consisting essentially of, orconsisting of the following polypeptide sequences: X₄X₅RKH (SEQ IDNO:324), X₄X₅RRR (SEQ ID NO:325), X₄X₅KKK (SEQ ID NO:326), X₄X₅HHH (SEQID NO:327), X₄X₅RKK (SEQ ID NO:328), X₄X₅RKR (SEQ ID NO:329), X₄X₅KKH(SEQ ID NO:330), X₄X₅HKH (SEQ ID NO:331), X₄X₅RRH (SEQ ID NO:332) andX₄X₅RHH (SEQ ID NO:333) where X₄ is any amino acid and X₅ is any aminoacid.

In other embodiments Sp35 peptide fragments to which certain antibodies,or antigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, an Sp35 polypeptide comprising,consisting essentially of, or consisting of peptides of the Ig domain ofSp35 or fragments, variants, or derivatives of such polypeptides.Specifically, polypeptides comprising, consisting essentially of, orconsisting of the following polypeptide sequences: ITX₆X₇X₈ (SEQ IDNO:334), ACX₆X₇X₈ (SEQ ID NO:335), VCX₆X₇X₈ (SEQ ID NO:336) and SPX₆X₇X₈(SEQ ID NO:337) where X₆ is lysine, arginine, histidine, glutamine, orasparagine, X₇ is any amino acid and X₈ is lysine, arginine, histidine,glutamine, or asparagine. For example, a polypeptide comprising,consisting essentially of, or consisting of the following polypeptidesequence: SPRLH (SEQ ID NO:338).

Sp35 peptide fragments to which certain antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the present inventionbind include, an Sp35 polypeptide comprising, consisting essentially of,or consisting of peptides which contain amino acids 452-458 in the Igdomain of Sp35, or derivatives thereof, wherein amino acid 452 is atryptophan or phenylalanine residue.

Additional Sp35 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, an Sp35 polypeptide comprising,consisting essentially of, or consisting of peptides of the basic domainof Sp35. Specifically, peptides comprising, consisting essentially of,or consisting of the following polypeptide sequences: RRARIRDRK (SEQ IDNO:339), KKVKVKEKR (SEQ ID NO:340), RRLRLRDRK (SEQ ID NO:341), RRGRGRDRK(SEQ ID NO:342) and RRIRARDRK (SEQ ID NO:343).

Additional exemplary soluble Sp35 polypeptides and methods and materialsfor obtaining these molecules for producing antibodies or antibodyfragments of the present invention may be found, e.g., in InternationalPatent Application No. PCT/US2004/008323, incorporated herein byreference in its entirety.

Methods of making antibodies are well known in the art and describedherein. Once antibodies to various fragments of, or to the full-lengthSp35 without the signal sequence, have been produced, determining whichamino acids, or epitope, of Sp35 to which the antibody or antigenbinding fragment binds can be determined by eptiope mapping protocols asdescribed herein as well as methods known in the art (e.g. doubleantibody-sandwich ELISA as described in “Chapter 11—Immunology,” CurrentProtocols in Molecular Biology, Ed. Ausubel et al., v.2, John Wiley &Sons, Inc. (1996)). Additional epitope mapping protocols may be found inMorris, G. Epitope Mapping Protocols, New Jersey: Humana Press (1996),which are both incorporated herein by reference in their entireties.Epitope mapping can also be performed by commercially available means(i.e. ProtoPROBE, Inc. (Milwaukee, Wis.)).

Additionally, antibodies produced which bind to any portion of Sp35 canthen be screened for their ability to act as an antagonist of Sp35 andthus promote neurite outgrowth, neuronal and oligodendrocyte survival,proliferation and differentiation as well as promote myelination.Antibodies can be screened for oligodendrocyte/neuronal survival byusing the method as described in Examples 10 and 11. Additionally,antibodies can be screened for their ability to promote myelination byusing the method of Example 9. Finally, antibodies can be screened fortheir ability to promote oligodendrocyte proliferation anddifferentiation, as well as neurite outgrowth by using the method asdescribed in Example 7. Other antagonist functions of antibodies of thepresent invention can be tested using other assays as described in theExamples herein.

In other embodiments, the present invention includes an antibody, orantigen-binding fragment, variant, or derivative thereof whichspecifically or preferentially binds to at least one epitope of Sp35,where the epitope comprises, consists essentially of, or consists of atleast about four to five amino acids of SEQ ID NO:2, at least seven, atleast nine, or between at least about 15 to about 30 amino acids of SEQID NO:2. The amino acids of a given epitope of SEQ ID NO:2 as describedmay be, but need not be contiguous or linear. In certain embodiments,the at least one epitope of Sp35 comprises, consists essentially of, orconsists of a non-linear epitope formed by the extracellular domain ofSp35 as expressed on the surface of a cell or as a soluble fragment,e.g., fused to an IgG Fc region. Thus, in certain embodiments the atleast one epitope of Sp35 comprises, consists essentially of, orconsists of at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 15, at least 20, at least 25, betweenabout 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguousamino acids of SEQ ID NO:2, where the non-contiguous amino acids form anepitope through protein folding.

In other embodiments, the present invention includes an antibody, orantigen-binding fragment, variant, or derivative thereof whichspecifically or preferentially binds to at least one epitope of Sp35,where the epitope comprises, consists essentially of, or consists of, inaddition to one, two, three, four, five, six or more contiguous ornon-contiguous amino acids of SEQ ID NO:2 as described above, and anadditional moiety which modifies the protein, e.g., a carbohydratemoiety may be included such that the Sp35 antibody binds with higheraffinity to modified target protein than it does to an unmodifiedversion of the protein. Alternatively, the Sp35 antibody does not bindthe unmodified version of the target protein at all.

In certain aspects, the present invention is directed to an antibody, orantigen-binding fragment, variant, or derivative thereof whichspecifically binds to a Sp35 polypeptide or fragment thereof, or an Sp35variant polypeptide, with an affinity characterized by a dissociationconstant (K_(D)) which is less than the K_(D) for said referencemonoclonal antibody.

In certain embodiments, an antibody, or antigen-binding fragment,variant, or derivative thereof of the invention binds specifically to atleast one epitope of Sp35 or fragment or variant described above, i.e.,binds to such an epitope more readily than it would bind to anunrelated, or random epitope; binds preferentially to at least oneepitope of Sp35 or fragment or variant described above, i.e., binds tosuch an epitope more readily than it would bind to a related, similar,homologous, or analogous epitope; competitively inhibits binding of areference antibody which itself binds specifically or preferentially toa certain epitope of Sp35 or fragment or variant described above; orbinds to at least one epitope of Sp35 or fragment or variant describedabove with an affinity characterized by a dissociation constant K_(D) ofless than about 5×10⁻² M, about 10⁻² M, about 5×10⁻³ M, about 10⁻³ M,about 5×10⁻⁴ M, about 10⁻⁴ M, about 5×10⁻⁵ M, about 10⁻⁵ M, about 5×10⁻⁶M, about 10⁻⁶ M, about 5×10⁻⁷ M, about 10⁻⁷ M, about 5×10⁻⁸ M, about10⁻⁸ M, about 5×10⁻⁹ M, about 10⁻⁹ M, about 5×10⁻¹⁰ M, about 10⁻¹⁰ M,about 5×10⁻¹¹ M, about 10⁻¹¹ M, about 5×10⁻¹² M, about 10⁻¹² M, about5×10⁻¹³ M, about 10⁻¹³ M, about 5×10⁻¹⁴ M, about 10⁻¹⁴ M, about 5×10⁻¹⁵M, or about 10⁻¹⁵ M. In a particular aspect, the antibody or fragmentthereof preferentially binds to a human Sp35 polypeptide or fragmentthereof, relative to a murine Sp35 polypeptide or fragment thereof.

As used in the context of antibody binding dissociation constants, theterm “about” allows for the degree of variation inherent in the methodsutilized for measuring antibody affinity. For example, depending on thelevel of precision of the instrumentation used, standard error based onthe number of samples measured, and rounding error, the term “about 10⁻²M” might include, for example, from 0.05 M to 0.005 M.

In specific embodiments, an antibody, or antigen-binding fragment,variant, or derivative thereof of the invention binds Sp35 polypeptidesor fragments or variants thereof with an off rate (k(off)) of less thanor equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³ sec⁻¹.Alternatively, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention binds Sp35 polypeptides or fragmentsor variants thereof with an off rate (k(off)) of less than or equal to5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹.

In other embodiments, an antibody, or antigen-binding fragment, variant,or derivative thereof of the invention binds Sp35 polypeptides orfragments or variants thereof with an on rate (k(on)) of greater than orequal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹sec⁻¹. Alternatively, an antibody, or antigen-binding fragment, variant,or derivative thereof of the invention binds Sp35 polypeptides orfragments or variants thereof with an on rate (k(on)) greater than orequal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×106 M⁻¹sec⁻¹ or 10⁷ M⁻¹ sec⁻¹.

In various embodiments, an Sp35 antibody, or antigen-binding fragment,variant, or derivative thereof as described herein is an antagonist ofSp35 activity. In certain embodiments, for example, binding of anantagonist Sp35 antibody to Sp35, as expressed on neurons, blocksmyelin-associated neurite outgrowth inhibition or neuronal cell death.In other embodiments, binding of the Sp35 antibody to Sp35, as expressedon oligodendrocytes, blocks inhibition of oligodendrocyte growth ordifferentiation, or blocks demyelination or dysmyelination of CNSneurons.

Unless it is specifically noted, as used herein a “fragment thereof” inreference to an antibody refers to an antigen-binding fragment, i.e., aportion of the antibody which specifically binds to the antigen. In oneembodiment, an Sp35 antibody, e.g., an antibody of the invention is abispecific Sp35 antibody, binding polypeptide, or antibody, e.g., abispecific antibody, minibody, domain deleted antibody, or fusionprotein having binding specificity for more than one epitope, e.g., morethan one antigen or more than one epitope on the same antigen. In oneembodiment, a bispecific Sp35 antibody, binding polypeptide, or antibodyhas at least one binding domain specific for at least one epitope on atarget polypeptide disclosed herein, e.g., Sp35. In another embodiment,a bispecific Sp35 antibody, binding polypeptide, or antibody has atleast one binding domain specific for an epitope on a target polypeptideand at least one target binding domain specific for a drug or toxin. Inyet another embodiment, a bispecific Sp35 antibody, binding polypeptide,or antibody has at least one binding domain specific for an epitope on atarget polypeptide disclosed herein, and at least one binding domainspecific for a prodrug. A bispecific Sp35 antibody, binding polypeptide,or antibody may be a tetravalent antibody that has two target bindingdomains specific for an epitope of a target polypeptide disclosed hereinand two target binding domains specific for a second target. Thus, atetravalent bispecific Sp35 antibody, binding polypeptide, or antibodymay be bivalent for each specificity.

Sp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention, as known by those of ordinary skill in theart, can comprise a constant region which mediates one or more effectorfunctions. For example, binding of the C1 component of complement to anantibody constant region may activate the complement system. Activationof complement is important in the opsonisation and lysis of cellpathogens. The activation of complement also stimulates the inflammatoryresponse and may also be involved in autoimmune hypersensitivity.Further, antibodies bind to receptors on various cells via the Fcregion, with a Fc receptor binding site on the antibody Fc regionbinding to a Fc receptor (FcR) on a cell. There are a number of Fcreceptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production.

Accordingly, certain embodiments of the invention include an Sp35antibody, or antigen-binding fragment, variant, or derivative thereof,in which at least a fraction of one or more of the constant regiondomains has been deleted or otherwise altered so as to provide desiredbiochemical characteristics such as reduced effector functions, theability to non-covalently dimerize, increased ability to localize at thesite of a tumor, reduced serum half-life, or increased serum half-lifewhen compared with a whole, unaltered antibody of approximately the sameimmunogenicity. For example, certain antibodies for use in thediagnostic and treatment methods described herein are domain deletedantibodies which comprise a polypeptide chain similar to animmunoglobulin heavy chain, but which lack at least a portion of one ormore heavy chain domains. For instance, in certain antibodies, oneentire domain of the constant region of the modified antibody will bedeleted, for example, all or part of the C_(H)2 domain will be deleted.

In certain Sp35 antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as tumorlocalization, biodistribution and serum half-life, may easily bemeasured and quantified using well know immunological techniques withoutundue experimentation.

Modified forms of Sp35 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can be made from wholeprecursor or parent antibodies using techniques known in the art.Exemplary techniques are discussed in more detail herein.

In certain embodiments both the variable and constant regions of Sp35antibodies, or antigen-binding fragments, variants, or derivativesthereof are fully human. Fully human antibodies can be made usingtechniques that are known in the art and as described herein. Forexample, fully human antibodies against a specific antigen can beprepared by administering the antigen to a transgenic animal which hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled. Exemplarytechniques that can be used to make such antibodies are described inU.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140 which are incorporated byreference in their entireties. Other techniques are known in the art.Fully human antibodies can likewise be produced by various displaytechnologies, e.g., phage display or other viral display systems, asdescribed in more detail elsewhere herein.

Sp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be made or manufactured using techniquesthat are known in the art. In certain embodiments, antibody molecules orfragments thereof are “recombinantly produced,” i.e., are produced usingrecombinant DNA technology. Exemplary techniques for making antibodymolecules or fragments thereof are discussed in more detail elsewhereherein.

Sp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention also include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromspecifically binding to its cognate epitope. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

In certain embodiments, Sp35 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention will not elicit adeleterious immune response in the animal to be treated, e.g., in ahuman. In one embodiment, Sp35 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention are modified to reducetheir immunogenicity using art-recognized techniques. For example,antibodies can be humanized, primatized, deimmunized, or chimericantibodies can be made. These types of antibodies are derived from anon-human antibody, typically a murine or primate antibody, that retainsor substantially retains the antigen-binding properties of the parentantibody, but which is less immunogenic in humans. This may be achievedby various methods, including (a) grafting the entire non-human variabledomains onto human constant regions to generate chimeric antibodies; (b)grafting at least a part of one or more of the non-human complementaritydetermining regions (CDRs) into a human framework and constant regionswith or without retention of critical framework residues; or (c)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like section by replacement of surface residues. Suchmethods are disclosed in Morrison et al., Proc. Natl. Acad. Sci.81:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988);Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun.28:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), and U.S.Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all of whichare hereby incorporated by reference in their entirety.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes (see, e.g., WO9852976A1,WO0034317A2). For example, V_(H) and V_(L) sequences from the startingantibody are analyzed and a human T cell epitope “map” from each Vregion showing the location of epitopes in relation tocomplementarity-determining regions (CDRs) and other key residues withinthe sequence. Individual T cell epitopes from the T cell epitope map areanalyzed in order to identify alternative amino acid substitutions witha low risk of altering activity of the final antibody. A range ofalternative V_(H) and V_(L) sequences are designed comprisingcombinations of amino acid substitutions and these sequences aresubsequently incorporated into a range of binding polypeptides, e.g.,Sp35-specific antibodies or immunospecific fragments thereof for use inthe diagnostic and treatment methods disclosed herein, which are thentested for function. Typically, between 12 and 24 variant antibodies aregenerated and tested. Complete heavy and light chain genes comprisingmodified V and human C regions are then cloned into expression vectorsand the subsequent plasmids introduced into cell lines for theproduction of whole antibody. The antibodies are then compared inappropriate biochemical and biological assays, and the optimal variantis identified.

Sp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention may be generated by any suitable method knownin the art. Polyclonal antibodies to an antigen of interest can beproduced by various procedures well known in the art. For example, anSp35 antibody, e.g., a binding polypeptide, e.g., an Sp35-specificantibody or immunospecific fragment thereof can be administered tovarious host animals including, but not limited to, rabbits, mice, rats,chickens, hamsters, goats, donkeys, etc., to induce the production ofsera containing polyclonal antibodies specific for the antigen. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and include but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants arealso well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas Elsevier, N.Y., 563-681 (1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Thus, the term“monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. Monoclonal antibodies can be prepared using Sp35knockout mice to increase the regions of epitope recognition. Monoclonalantibodies can be prepared using a wide variety of techniques known inthe art including the use of hybridoma and recombinant and phage displaytechnology as described elsewhere herein.

Using art recognized protocols, in one example, antibodies are raised inmammals by multiple subcutaneous or intraperitoneal injections of therelevant antigen (e.g., purified tumor associated antigens such as Sp35or cells or cellular extracts comprising such antigens) and an adjuvant.This immunization typically elicits an immune response that comprisesproduction of antigen-reactive antibodies from activated splenocytes orlymphocytes. While the resulting antibodies may be harvested from theserum of the animal to provide polyclonal preparations, it is oftendesirable to isolate individual lymphocytes from the spleen, lymph nodesor peripheral blood to provide homogenous preparations of monoclonalantibodies (MAbs). Preferably, the lymphocytes are obtained from thespleen.

In this well known process (Kohler et al., Nature 256:495 (1975)) therelatively short-lived, or mortal, lymphocytes from a mammal which hasbeen injected with antigen are fused with an immortal tumor cell line(e.g. a myeloma cell line), thus, producing hybrid cells or “hybridomas”which are both immortal and capable of producing the genetically codedantibody of the B cell. The resulting hybrids are segregated into singlegenetic strains by selection, dilution, and regrowth with eachindividual strain comprising specific genes for the formation of asingle antibody. They produce antibodies which are homogeneous against adesired antigen and, in reference to their pure genetic parentage, aretermed “monoclonal.”

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. Those skilledin the art will appreciate that reagents, cell lines and media for theformation, selection and growth of hybridomas are commercially availablefrom a number of sources and standardized protocols are wellestablished. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production of monoclonal antibodies against thedesired antigen. Preferably, the binding specificity of the monoclonalantibodies produced by hybridoma cells is determined by in vitro assayssuch as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). After hybridoma cells are identified thatproduce antibodies of the desired specificity, affinity and/or activity,the clones may be subcloned by limiting dilution procedures and grown bystandard methods (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, pp 59-103 (1986)). It will further beappreciated that the monoclonal antibodies secreted by the subclones maybe separated from culture medium, ascites fluid or serum by conventionalpurification procedures such as, for example, protein-A, hydroxylapatitechromatography, gel electrophoresis, dialysis or affinitychromatography.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedby proteolytic cleavage of immunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the variable region, the lightchain constant region and the C_(H)1 domain of the heavy chain.

Those skilled in the art will also appreciate that DNA encodingantibodies or antibody fragments (e.g., antigen binding sites) may alsobe derived from antibody libraries, such as phage display libraries. Ina particular, such phage can be utilized to display antigen-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an antigen binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv OE DAB (individual Fv region from light or heavychains) or disulfide stabilized Fv antibody domains recombinantly fusedto either the phage gene III or gene VIII protein. Exemplary methods areset forth, for example, in EP 368 684 B1; U.S. Pat. No. 5,969,108,Hoogenboom, H. R. and Chames, Immunol. Today 21:371 (2000); Nagy et al.Nat. Med. 8:801 (2002); Huie et al., Proc. Natl. Acad. Sci. USA 98:2682(2001); Lui et al., J. Mol. Biol. 315:1063 (2002), each of which isincorporated herein by reference. Several publications (e.g., Marks etal., Bio/Technology 10:779-783 (1992)) have described the production ofhigh affinity human antibodies by chain shuffling, as well ascombinatorial infection and in vivo recombination as a strategy forconstructing large phage libraries. In another embodiment, Ribosomaldisplay can be used to replace bacteriophage as the display platform(see, e.g., Hanes et al., Nat. Biotechnol. 18:1287 (2000); Wilson etal., Proc. Natl. Acad. Sci. USA 98:3750 (2001); or Irving et al., J.Immunol. Methods 248:31 (2001)). In yet another embodiment, cell surfacelibraries can be screened for antibodies (Boder et al., Proc. Natl.Acad. Sci. USA 97:10701 (2000); Daugherty et al., J. Immunol. Methods243:211 (2000)). Such procedures provide alternatives to traditionalhybridoma techniques for the isolation and subsequent cloning ofmonoclonal antibodies.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. For example, DNA sequences encoding V_(H) and V_(L)regions are amplified from animal cDNA libraries (e.g., human or murinecDNA libraries of lymphoid tissues) or synthetic cDNA libraries. Incertain embodiments, the DNA encoding the V_(H) and V_(L) regions arejoined together by an scFv linker by PCR and cloned into a phagemidvector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is electroporatedin E. coli and the E. coli is infected with helper phage. Phage used inthese methods are typically filamentous phage including fd and M13 andthe V_(H) or V_(L) regions are usually recombinantly fused to either thephage gene III or gene VIII. Phage expressing an antigen binding domainthat binds to an antigen of interest (i.e., an Sp35 polypeptide or afragment thereof) can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead.

Additional examples of phage display methods that can be used to makethe antibodies include those disclosed in Brinkman et al., J. Immunol.Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186(1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persicet al., Gene 187:9-18 (1997); Burton et al., Advances in Immunology57:191-280 (1994); PCT Application No. PCT/GB91/01134; PCT publicationsWO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)₂ fragments can also be employed using methods known in the artsuch as those disclosed in PCT publication WO 92/22324; Mullinax et al.,BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34(1995); and Better et al., Science 240:1041-1043 (1988) (said referencesincorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entireties. Humanized antibodies areantibody molecules derived from a non-human species antibody that bindthe desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332, which is incorporated by reference in its entirety).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a desired target polypeptide. Monoclonal antibodies directedagainst the antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B-celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. For an overviewof this technology for producing human antibodies, see Lonberg andHuszar Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/Technology 12:899-903(1988). See also, U.S. Pat. No. 5,565,332, which is incorporated byreference in its entirety.)

Further, antibodies to target polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”target polypeptides using techniques well known to those skilled in theart. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444 (1989) andNissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodieswhich bind to and competitively inhibit polypeptide multimerizationand/or binding of a polypeptide of the invention to a ligand can be usedto generate anti-idiotypes that “mimic” the polypeptide multimerizationand/or binding domain and, as a consequence, bind to and neutralizepolypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fabfragments of such anti-idiotypes can be used in therapeutic regimens toneutralize polypeptide ligand. For example, such anti-idiotypicantibodies can be used to bind a desired target polypeptide and/or tobind its ligands/receptors, and thereby block its biological activity.

In another embodiment, DNA encoding desired monoclonal antibodies may bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Theisolated and subcloned hybridoma cells serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transfected into prokaryotic or eukaryotic host cellssuch as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO)cells or myeloma cells that do not otherwise produce immunoglobulins.More particularly, the isolated DNA (which may be synthetic as describedherein) may be used to clone constant and variable region sequences forthe manufacture antibodies as described in Newman et al., U.S. Pat. No.5,658,570, filed Jan. 25, 1995, which is incorporated by referenceherein. Essentially, this entails extraction of RNA from the selectedcells, conversion to cDNA, and amplification by PCR using Ig specificprimers. Suitable primers for this purpose are also described in U.S.Pat. No. 5,658,570. As will be discussed in more detail below,transformed cells expressing the desired antibody may be grown up inrelatively large quantities to provide clinical and commercial suppliesof the immunoglobulin.

In one embodiment, an Sp35 antibody of the invention comprises at leastone heavy or light chain CDR of an antibody molecule. In anotherembodiment, an Sp35 antibody of the invention comprises at least twoCDRs from one or more antibody molecules. In another embodiment, an Sp35antibody of the invention comprises at least three CDRs from one or moreantibody molecules. In another embodiment, an Sp35 antibody of theinvention comprises at least four CDRs from one or more antibodymolecules. In another embodiment, an Sp35 antibody of the inventioncomprises at least five CDRs from one or more antibody molecules. Inanother embodiment, an Sp35 antibody of the invention comprises at leastsix CDRs from one or more antibody molecules. Exemplary antibodymolecules comprising at least one CDR that can be included in thesubject Sp35 antibodies are described herein.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody. Theframework regions may be naturally occurring or consensus frameworkregions, and preferably human framework regions (see, e.g., Chothia etal., J. Mol. Biol. 278:457-479 (1998) for a listing of human frameworkregions). Preferably, the polynucleotide generated by the combination ofthe framework regions and CDRs encodes an antibody that specificallybinds to at least one epitope of a desired polypeptide, e.g., Sp35.Preferably, one or more amino acid substitutions may be made within theframework regions, and, preferably, the amino acid substitutions improvebinding of the antibody to its antigen. Additionally, such methods maybe used to make amino acid substitutions or deletions of one or morevariable region cysteine residues participating in an intrachaindisulfide bond to generate antibody molecules lacking one or moreintrachain disulfide bonds. Other alterations to the polynucleotide areencompassed by the present invention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asused herein, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-554 (1989)) can be adapted to produce singlechain antibodies. Single chain antibodies are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain antibody. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

Yet other embodiments of the present invention comprise the generationof human or substantially human antibodies in transgenic animals (e.g.,mice) that are incapable of endogenous immunoglobulin production (seee.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 eachof which is incorporated herein by reference). For example, it has beendescribed that the homozygous deletion of the antibody heavy-chainjoining region in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of a humanimmunoglobulin gene array to such germ line mutant mice will result inthe production of human antibodies upon antigen challenge. Anotherpreferred means of generating human antibodies using SCID mice isdisclosed in U.S. Pat. No. 5,811,524 which is incorporated herein byreference. It will be appreciated that the genetic material associatedwith these human antibodies may also be isolated and manipulated asdescribed herein.

Yet another highly efficient means for generating recombinant antibodiesis disclosed by Newman, Biotechnology 10: 1455-1460 (1992).Specifically, this technique results in the generation of primatizedantibodies that contain monkey variable domains and human constantsequences. This reference is incorporated by reference in its entiretyherein. Moreover, this technique is also described in commonly assignedU.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which isincorporated herein by reference.

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized mammal and culturedfor about 7 days in vitro. The cultures can be screened for specificIgGs that meet the screening criteria. Cells from positive wells can beisolated. Individual Ig-producing B cells can be isolated by FACS or byidentifying them in a complement-mediated hemolytic plaque assay.Ig-producing B cells can be micromanipulated into a tube and the V_(H)and V_(L) genes can be amplified using, e.g., RT-PCR. The V_(H) andV_(L) genes can be cloned into an antibody expression vector andtransfected into cells (e.g., eukaryotic or prokaryotic cells) forexpression.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Antibodies for use in the diagnostic and therapeutic methods disclosedherein can be produced by any method known in the art for the synthesisof antibodies, in particular, by chemical synthesis or preferably, byrecombinant expression techniques as described herein.

In one embodiment, an Sp35 antibody, or antigen-binding fragment,variant, or derivative thereof of the invention comprises a syntheticconstant region wherein one or more domains are partially or entirelydeleted (“domain-deleted antibodies”). In certain embodiments compatiblemodified antibodies will comprise domain deleted constructs or variantswherein the entire C_(H)2 domain has been removed (ΔC_(H)2 constructs).For other embodiments a short connecting peptide may be substituted forthe deleted domain to provide flexibility and freedom of movement forthe variable region. Those skilled in the art will appreciate that suchconstructs are particularly preferred due to the regulatory propertiesof the C_(H)2 domain on the catabolic rate of the antibody. Domaindeleted constructs can be derived using a vector (e.g., from Biogen IDECIncorporated) encoding an IgG₁ human constant domain (see, e.g., WO02/060955A2 and WO02/096948A2, which are incorporated by reference intheir entireties). This exemplary vector was engineered to delete theC_(H)2 domain and provide a synthetic vector expressing a domain deletedIgG₁ constant region.

In certain embodiments, Sp35 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention are minibodies.Minibodies can be made using methods described in the art (see, e.g.,see e.g., U.S. Pat. No. 5,837,821 or WO 94/09817A1, which areincorporated by reference in their entireties).

In one embodiment, an Sp35 antibody, or antigen-binding fragment,variant, or derivative thereof of the invention comprises animmunoglobulin heavy chain having deletion or substitution of a few oreven a single amino acid as long as it permits association between themonomeric subunits. For example, the mutation of a single amino acid inselected areas of the C_(H)2 domain may be enough to substantiallyreduce Fc binding and thereby increase tumor localization. Similarly, itmay be desirable to simply delete that part of one or more constantregion domains that control the effector function (e.g. complementbinding) to be modulated. Such partial deletions of the constant regionsmay improve selected characteristics of the antibody (serum half-life)while leaving other desirable functions associated with the subjectconstant region domain intact. Moreover, as alluded to above, theconstant regions of the disclosed antibodies may be synthetic throughthe mutation or substitution of one or more amino acids that enhancesthe profile of the resulting construct. In this respect it may bepossible to disrupt the activity provided by a conserved binding site(e.g. Fc binding) while substantially maintaining the configuration andimmunogenic profile of the modified antibody. Yet other embodimentscomprise the addition of one or more amino acids to the constant regionto enhance desirable characteristics such as effector function orprovide for more cytotoxin or carbohydrate attachment. In suchembodiments it may be desirable to insert or replicate specificsequences derived from selected constant region domains.

The present invention also provides antibodies that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the V_(H) regions and/or V_(L) regions)described herein, which antibodies or fragments thereofimmunospecifically bind to an Sp35 polypeptide or fragment or variantthereof. Standard techniques known to those of skill in the art can beused to introduce mutations in the nucleotide sequence encoding an Sp35antibody, including, but not limited to, site-directed mutagenesis andPCR-mediated mutagenesis which result in amino acid substitutions.Preferably, the variants (including derivatives) encode less than 50amino acid substitutions, less than 40 amino acid substitutions, lessthan 30 amino acid substitutions, less than 25 amino acid substitutions,less than 20 amino acid substitutions, less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the reference V_(H) region, V_(H)CDR1, V_(H)CDR2, V_(H)CDR3,V_(L) region, V_(L)CDR1, V_(L)CDR2, or V_(L)CDR3. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a side chain with a similar charge.Families of amino acid residues having side chains with similar chargeshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Alternatively, mutations can be introduced randomly alongall or part of the coding sequence, such as by saturation mutagenesis,and the resultant mutants can be screened for biological activity toidentify mutants that retain activity (e.g., the ability to bind an Sp35polypeptide).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g.,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein may routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to immunospecifically bind at least oneepitope of an Sp35 polypeptide) can be determined using techniquesdescribed herein or by routinely modifying techniques known in the art.

IV. Polynucleotides Encoding Sp35 Antibodies

The present invention also provides for nucleic acid molecules encodingSp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region(V_(H)), where at least one of the CDRs of the heavy chain variableregion or at least two of the CDRs of the heavy chain variable regionare at least 80%, 85%, 90% or 95% identical to reference heavy chainCDR1, CDR2, or CDR3 amino acid sequences from monoclonal Sp35 antibodiesdisclosed herein. Alternatively, the CDR1, CDR2, and CDR3 regions of theV_(H) are at least 80%, 85%, 90% or 95% identical to reference heavychain CDR1, CDR2, and CDR3 amino acid sequences from monoclonal Sp35antibodies disclosed herein. Thus, according to this embodiment a heavychain variable region of the invention has CDR1, CDR2, or CDR3polypeptide sequences related to the polypeptide sequences shown inTable 4:

TABLE 4 Reference VH CDR1, CDR2, and CDR3 amino acid sequences* AntibodyName VH-CDR1 VH-CDR2 VH-CDR3 Li10 P = TYPMV P = WIGPSGGVTAYADSVKG P =PYSSGWWDFDL (SEQ ID NO: 6) (SEQ ID NO: 8) (SEQ ID NO: 10) N =ACTTACCCTATGGTT N = TGGATCGGTCCTTCT N = CCCTATAGCAGTGGCT (SEQ ID NO: 5)GGTGGCGTTACTGCTTA GGTGGGACTTCGATCTC TGCTGACTCCGTTAAAGGT (SEQ ID NO: 9)(SEQ ID NO: 7) Li07 P = MYFMG P = SISPSGGFTSYADSVKG P = DRHAFDI(SEQ ID NO: 12) (SEQ ID NO: 14) (SEQ ID NO: 16) N = ATGTACTTTATGGGT N =TCTATCTCTCCTTCTGGTGGCTTTAC N = GATCGGCATGCTTTTGATATC (SEQ ID NO: 11)TTCTTATGCTGACTCCGTTAAAGGT (SEQ ID NO: 15) (SEQ ID NO: 13) Li05 P = AYAMGP = SIVSSGGYTDYADSVKG P = EGDHNAFDI (SEQ ID NO: 18) (SEQ ID NO: 20)(SEQ ID NO: 22) N = CTTACGCTATGGGT N = TCTATCGTTTCTTCTGGTGGCT N =GAGGGTGACCATAATGCTTTT (SEQ ID NO: 17) ATACTGATTATGCTGACTCCGTTAAAGGTGATATC (SEQ ID NO: 19) (SEQ ID NO: 21) Li11 P = SYAMY P =SISTSGGYTGYADSVKG P = DTSDNDYYYMDV (SEQ ID NO: 24) (SEQ ID NO: 26)(SEQ ID NO: 28) N = TCTTACGCTATGTAT N = TCTATCTCTACTTCTGGTGGCTA N =GATACCAGCGATAATGAC (SEQ ID NO: 23) TACTGGTTATGCTGACTCCGTTAAAGGTTACTACTACATGGACGTC (SEQ ID NO: 25) (SEQ ID NO: 27) Li01 P = KYQMT P =SIYPSGGNTVYADSVKG P = GTTEAVFDY (SEQ ID NO: 30) (SEQ ID NO: 32)(SEQ ID NO: 34) N == AAGTACCAGATGAC N = TCTATCTATCCTTCTGGTGGCAA N =GGGACTACAGAGGCAGTCTT (SEQ ID NO: 29) TACTGTTTATGCTGACTCCGTTAAAGGTTGACTAC (SEQ ID NO: 31) (SEQ ID NO: 33) Li12 P = QYNMF P =RISSSGGMTMYADSVKG P = EALRPYCSGGSCYSDYYYYGMDV (SEQ ID NO: 36)(SEQ ID NO: 38) (SEQ ID NO: 40) N = CAGTACAATATGTTT N =CGTATCTCTTCTTCTGGTGGCAT N = GAAGCGTTACGGCCTTATTG (SEQ ID NO: 35)GACTATGTATGCTGACTCCGTTAAAGGT TAGTGGTGGTAGCTGCTACTCCG (SEQ ID NO: 37)ACTACTACTACTACGGTATGGAC GTC (SEQ ID NO: 39) Li06 P = EYPMD P =SIYSSGGSTVYADSIKG P = EGDSDAFDI (SEQ ID NO: 42) (SEQ ID NO: 44)(SEQ ID NO: 46) N = GAGTACCCTATGGAT N = TCTATCTATTCTTCTGGTGGCTC N =GAGGGTGACTCTGATGCTTTT (SEQ ID NO: 41) TACTGTTTATGCTGACTCCATTAAAGGTGATATC (SEQ ID NO: 43) (SEQ ID NO: 45) Li08 P = HYEMV P =SIRSSGGATKYADSVKG P = ESPDDYFDY (SEQ ID NO: 48) (SEQ ID NO: 50)(SEQ ID NO: 52 N = CATTACGAGATGGTT N = TCTATCCGTTCTTCTGGTGGCGCTAC N =GAGTCGCCAGACGACTACTTT (SEQ ID NO: 47) TAAGTATGCTGACTCCGTTAAAGGT GACTAC(SEQ ID NO: 49) (SEQ ID NO: 51) Li03 P = QYPME P = GIYPSGGSTVYADSVKG P =AGQWLGDFDY (SEQ ID NO: 54) (SEQ ID NO: 56) (SEQ ID NO: 58) N =CAGTACCCTATGGAG N = GGTATCTATCCTTCTGGTGGCTCTA N = GCGGGGCAGTGGCTGGGGGAC(SEQ ID NO: 53) CTGTTTATGCTGACTCCGTTAAAGGT TTTGACTAC (SEQ ID NO: 55)(SEQ ID NO: 57) Li09 P = MYSMV P = YISPSGGKTMYADSVKG P =DSRRRYYDFWSGYHNYYYYYMDV (SEQ ID NO: 60) (SEQ ID NO: 62) (SEQ ID NO: 64)N = ATGTACTCTATGGTT N = TATATCTCTCCTTCTGGTGGCAAG N =GATTCGAGACGCCGGTATTACGA (SEQ ID NO: 59) ACTATGTATGCTGACTCCGTTAAAGGTTTTTTGGAGTGGTTATCACAACTACT (SEQ ID NO: 61) ACTACTACTACATGGACGTC(SEQ ID NO: 63) Li04 P = RYNMG P = VIYPSGGGTHYADSVKG P = SIADDAFDI(SEQ ID NO: 66) (SEQ ID NO: 68) (SEQ ID NO: 70) N = CGTTACAATATGGGT N =GTTATCTATCCTTCTGGTGGCGGT N = TCTATAGCAGATGATGCTTTTGATATC (SEQ ID NO: 65)ACTCATTATGCTGACTCCGTTAAAGGT (SEQ ID NO: 69) (SEQ ID NO: 67) Li02 P =TYEMI P = SIGPSGGLTWYADSVKG P = MYYCVRIDDSSGWAFDI (SEQ ID NO: 72)(SEQ ID NO: 74) (SEQ ID NO: 76) N = ACTTACGAGATGATT N =TCTATCGGTCCTTCTGGTGGCC N = ATGTATTACTGTGTACGGATTGATGA (SEQ ID NO: 71)TTACTTGGTATGCTGACTCCGTTAAA TAGTAGTGGTTGGGCTTTTGATATC (SEQ ID NO: 73)(SEQ ID NO: 75) Li13 P = HYEMY P = RIVSSGGFTKYADSVKG P = EGDNDAFDI(SEQ ID NO: 389) (SEQ ID NO: 390) (SEQ ID NO: 391) Li32 P = AYMMQ P =SISPSGGNTKYADSVKG P = GDYGYWFDP (SEQ ID NO: 395) (SEQ ID NO: 396)(SEQ ID NO: 397) Li33 P = IYPMF P = WIGPSGGITKYADSVKG P = EGHNDWYFDL(SEQ ID NO: 401) (SEQ ID NO: 402) (SEQ ID NO: 403) Li34 P = NYEMY P =GIYSSGGITVYADSVKG P = AAILDWYFDL (SEQ ID NO: 407) (SEQ ID NO: 408)(SEQ ID NO: 409) 1A47 P = NYGMN P = WINTDTGEPTYTEDFQG P = EGVHFDY(SEQ ID NO: 77) (SEQ ID NO: 78) (SEQ ID NO: 79) 2F3 P = FSDAWLD P =EIRSKANNHATNYAESVKG P = SFAY (SEQ ID NO: 80) (SEQ ID NO: 81)(SEQ ID NO: 82) 3P1D10.2C3 P = SSWTQ P = AIYPGDGDTRYTQKFKG P = HNSYGMDYand (SEQ ID NO: 83) (SEQ ID NO: 84) (SEQ ID NO: 85) 3P1E11.3B7 L1a.01P = GYSFTNYWIG P = IIDPDDSYTTYSPSFQG P = AEFYWGAYDG (SEQ ID NO: 195)(SEQ ID NO: 196) (SEQ ID NO: 197) L1a.02 P = GGSIRGNYWS P =SINYSGFTNPSLKG P = VRHWYFDV (SEQ ID NO: 198) (SEQ ID NO: 199)(SEQ ID NO: 200) L1a.03 P = GYTFNGFDMH P = WIDPYNGSTFYAQKFQG P =DFYMDGHYYIFDV (SEQ ID NO: 201) (SEQ ID NO: 202) (SEQ ID NO: 203) L1a.04P = GYSFSNYYIH P = IIDPGDSFTSYSPSFQG P = DLAWIDYGFDY (SEQ ID NO: 204)(SEQ ID NO: 205) (SEQ ID NO: 206) L1a.05 P = GFTFTSHITVS P =SITGNGSTTYYADSVKG P = FYGDFDS (SEQ ID NO: 207) (SEQ ID NO: 208)(SEQ ID NO: 209) L1a.06 P = GFTFSSNWMS P = TIFYSGSSTYYADSVKG P =DLPMKGFIQQRYGFDDV (SEQ ID NO: 210) (SEQ ID NO: 211) (SEQ ID NO: 212)L1a.07 P = GFTFSGYAIS P = TIWGSGSTTYYADSVKG P = EYWYYDQFTAV(SEQ ID NO: 213) (SEQ ID NO: 214) (SEQ ID NO: 215) L1a.08 P =GDSVSSNSAAWS P = RIYYRSKWYNDYAVSVKS P = EVYSAGIMDY (SEQ ID NO: 216)(SEQ ID NO: 217) (SEQ ID NO: 218) L1a.09 P = GYSFTNHWIG P =IIDPSDSDTNYSPSFQG P = GFYGIADTFDV (SEQ ID NO: 219) (SEQ ID NO: 220)(SEQ ID NO: 221) L1a.10 P = GYSFTNYWIA P = MIYPDDSNTNYSPSFQG P =TNYLGFYDS (SEQ ID NO: 222) (SEQ ID NO: 223) (SEQ ID NO: 224) L1a.11 P =GFTFSDYGIS P = NILYDGSETYYADSVKG P = GYPTDDYSFDI (SEQ ID NO: 225)(SEQ ID NO: 226) (SEQ ID NO: 227) L1a.12 P = GDSVSDNSAAWG P =RIYYRSKWYNDYAVSVKS P = GRHEYGGLGYAEAMDH (SEQ ID NO: 228)(SEQ ID NO: 229) (SEQ ID NO: 230) L1a.13 P = GFTFSSYAMS P =AISGSGGSTYYADSVKG P = HYTYMHFEDY (SEQ ID NO: 231) (SEQ ID NO: 232)(SEQ ID NO: 233) *Determined by the Kabat system (see supra). N =nucleotide sequence, P = polypeptide sequence.In certain embodiments, an antibody or antigen-binding fragmentcomprising the VH encoded by the polynucleotide specifically orpreferentially binds to Sp35.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region (VH)in which the CDR1, CDR2, and CDR3 regions have polypeptide sequenceswhich are identical to the CDR1, CDR2, and CDR3 groups shown in Table 4.In certain embodiments, an antibody or antigen-binding fragmentcomprising the VH encoded by the polynucleotide specifically orpreferentially binds to Sp35.

In a further aspect, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region (VH)in which the CDR1, CDR2, and CDR3 regions are encoded by nucleotidesequences which are identical to the nucleotide sequences which encodethe CDR1, CDR2, and CDR3 groups shown in Table 4. In certainembodiments, an antibody or antigen-binding fragment comprising the VHencoded by the polynucleotide specifically or preferentially binds toSp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VH encoded byone or more of the polynucleotides described above specifically orpreferentially binds to the same epitope as a monoclonal antibodyselected from the group consisting of: 201′, 3A3, 3A6, 1A7, 1G7, 2B10,2C11, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9,3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (L100), 38-D01(Li022), 35-E04 (Li033), 36-C09 (Li04), 30-A11 (Li05), 34-F02 (Li06),29-E07 (Li07), 34-G04 (Li08), 36-A12 (Li09), 28-D02 (Li10), 30-B01(Li11), 34-B03 (L12), Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495(L1a.2), 3563 (L1a.3), 3564 (L1a.4), 3565 (L1a.5), 3566 (L1a.6), 3567(L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570 (L1a.10), 3571 (L1a.11), 3582(L1a.12), and 1968 (L1a.13), or will competitively inhibit such amonoclonal antibody from binding to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VH encoded byone or more of the polynucleotides described above specifically orpreferentially binds to an Sp35 polypeptide or fragment thereof, or aSp35 variant polypeptide, with an affinity characterized by adissociation constant (K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M,10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region(VL), where at least one of the CDRs of the light chain variable regionor at least two of the CDRs of the light chain variable region are atleast 80%, 85%, 90% or 95% identical to reference light chain CDR1,CDR2, or CDR3 amino acid sequences from monoclonal Sp35 antibodiesdisclosed herein. Alternatively, the CDR1, CDR2, and CDR3 regions of theVL are at least 80%, 85%, 90% or 95% identical to reference light chainCDR1, CDR2, and CDR3 amino acid sequences from monoclonal Sp35antibodies disclosed herein. Thus, according to this embodiment a lightchain variable region of the invention has CDR1, CDR2, or CDR3polypeptide sequences related to the polypeptide sequences shown inTable 5:

TABLE 5 Reference VL CDR1, CDR2, and CDR3 amino acid sequences* AntibodyName VL-CDR1 VL-CDR2 VL-CDR3 Li10 P = RASQGIGNWLA P = AASSLES P =QQAQTFPLT (SEQ ID NO: 87) (SEQ ID NO: 89) (SEQ ID NO: 91) N =CGGGCGAGTCAGGG N = GCTGCATCCAGTTTGGAAAGT N = CAACAGGCTCAGACTATTGGCAACTGGTTAGCC (SEQ ID NO: 88) TTTCCCGCTCACC (SEQ ID NO: 86)(SEQ ID NO: 90) Li07 P = SGDQLGDKHVA P = LDIKRPA P = QAWDIKTV(SEQ ID NO: 93) (SEQ ID NO: 95) (SEQ ID NO: 97) N = TCTGGAGATCAGTTG N =CTAGACATTAAGAGGCCCGCA N = CAGGCGTGGGACATC GGTGACAAACATGTGGCT(SEQ ID NO: 94) AAGACGGTC (SEQ ID NO: 92) (SEQ ID NO: 96) Li05 P =GGDNIGSKSVH P = DDYDRPS P = QVRDSRTEERV (SEQ ID NO: 99) (SEQ ID NO: 101)(SEQ ID NO: 103) N = GGGGGAGACAACAT N = GATGATTATGACCGGCCCTCA N =CAGGTGAGGGACAGCCG TGGAAGTAAGAGTGTCCAC (SEQ ID NO: 100) TACTGAGGAACGGGTG(SEQ ID NO: 98) (SEQ ID NO: 102) Li11 P = RASQEIANYLA P = DTYTLQT P =QQADIFPLS (SEQ ID NO: 105) (SEQ ID NO: 107) (SEQ ID NO: 109) N =CGGGCGAGTCAGGAG N = GATACATACACTTTGCAGACT N = CAACAGGCTGACATTTTATTGCCAACTACTTAGCC (SEQ ID NO: 106) CCCGCTCTCT (SEQ ID NO: 104)(SEQ ID NO: 108) Li01 P = QASQDISNYLN P = DASNLET P = QQADRFPAVT(SEQ ID NO: 111) (SEQ ID NO: 113) (SEQ ID NO: 115) N = CAGGCGAGTCAGGAN = GATGCATCCAATTTGGAAACA N = CAACAGGCTGACAGGTTC CATTAGCAACTATTTAAAT(SEQ ID NO: 112) CCTGCGGTCACT (SEQ ID NO: 110) (SEQ ID NO: 114) Li06 P =RASQSISSWLA P = AASSLRT P = LQDYSYPLT (SEQ ID NO: 117) (SEQ ID NO: 119)(SEQ ID NO: 121) N = CGGGCCAGTCAGAGTA N = GCTGCATCCAGTTTACGAACT N =CTACAAGATTACAGTTAC TTAGTAGCTGGTTGGCC (SEQ ID NO: 118) CCTCTCACT(SEQ ID NO: 116) (SEQ ID NO: 120) Li08 P = QASQDISYYLN P = DVSNLQT P =QQSDNLPLT (SEQ ID NO: 123) (SEQ ID NO: 125) (SEQ ID NO: 127) N =CAGGCGAGTCAGGAC N = GATGTATCCAATTTGCAAACA N = CAACAGTCTGATAATTAGTTACTATTTAAAT (SEQ ID NO: 124) ATCTCCCTCTCACT (SEQ ID NO: 122)(SEQ ID NO: 126) Li03 P = RASQSISSYLN P = AASSLQS P = QQSYSTPWT(SEQ ID NO: 129) (SEQ ID NO: 131) (SEQ ID NO: 133) N = GGGCAAGTCAGAGCN = GCTGCATCCAGTTTGCAAAGT N = CAACAGAGTTACA ATTAGCAGCTATTTAAAT(SEQ ID NO: 130) GTACCCCGTGGACG (SEQ ID NO: 128) (SEQ ID NO: 132) Li09P = RASQSIDTYLN P = AASKLED P = QQSYSPPLT (SEQ ID NO: 135)(SEQ ID NO: 137) (SEQ ID NO: 139) N = CGCGCAAGTCAGAGC N =GCTGCATCCAAGTTGGAAGAC N = CAACAGAGTTACAG ATCGACACCTATTTAAAT(SEQ ID NO: 136) TCCCCCTCTCAC (SEQ ID NO: 134) (SEQ ID NO: 138) Li02 P =SGDKLGDKFAS P = QDRKRLS P = QAWDTNTVV (SEQ ID NO: 141) (SEQ ID NO: 143)(SEQ ID NO: 145) N = TCTGGAGATAAATTGG N = CAAGATAGGAAGCGTCTCTCA N =CAGGCGTGGGACA GGGATAAATTTGCTTCC (SEQ ID NO: 142) CCAACACTGTGGTC(SEQ ID NO: 140) (SEQ ID NO: 144) Li13 P = RASQSVSSYLA P = DASNRAT P =QQRSNWPMYT (SEQ ID NO: 386) (SEQ ID NO: 387) (SEQ ID NO: 388) Li32 P =QASQDISYYLN P = DAFILEG P = QQSDQLPVT (SEQ ID NO: 392) (SEQ ID NO: 393)(SEQ ID NO: 394) Li33 P = RASQSVSSYLA P = DASNRAT P = QQYDKWPLT(SEQ ID NO: 398) (SEQ ID NO: 399) (SEQ ID NO: 400) Li34 P = HASQDISNYLSP = DAFNLET P = PQHYDNLPFT (SEQ ID NO: 404) (SEQ ID NO: 405)(SEQ ID NO: 406) 1A47 P = SASSSVSYMH P = DTSKLAS P = QQWSSNPFT(SEQ ID NO: 146) (SEQ ID NO: 147) (SEQ ID NO: 148) 2F3 P = RASGNIYNYLAP = NAKTLPD P = QHFWAIPYT (SEQ ID NO: 149) (SEQ ID NO: 150)(SEQ ID NO: 151) 3P1D10.2C3 P = KSSQSLLNSGNQKNYLT P = WASTRES P =QNDYSYPLFT (SEQ ID NO: 152) (SEQ ID NO: 153) (SEQ ID NO: 154) 3P1E11.3B7P = KSSQSLLNSGNQKSYLT P = WASTRES P = QNDYSYPLFT (SEQ ID NO: 155)(SEQ ID NO: 156) (SEQ ID NO: 157) L1a.01 P = SGDSLPSKFVH P = RDNNRPS P =SSYDALTD (SEQ ID NO: 234) (SEQ ID NO: 235) (SEQ ID NO: 236) L1a.02 P =RASQSITNSYLG P = DASSRAT P = QQASDAPE (SEQ ID NO: 237) (SEQ ID NO: 238)(SEQ ID NO: 239) L1a.03 P = RASQGINFWLN P = AGSNLQS P = MQDSDFPF(SEQ ID NO: 240) (SEQ ID NO: 241) (SEQ ID NO: 242) L1a.04 P =TGSSSNIGAGYDVS P = RNNNRPS P = QTYDNSTD (SEQ ID NO: 243)(SEQ ID NO: 244) (SEQ ID NO: 245) L1a.05 P = SGDNIRSYYVH P = EDSNRPS P =QSYDSAILLH (SEQ ID NO: 246) (SEQ ID NO: 247) (SEQ ID NO: 248) L1a.06 P =RSSQSLVLRTGYTYLN P = LVSNRAS P = QQYYGMPL (SEQ ID NO: 249)(SEQ ID NO: 250) (SEQ ID NO: 251) L1a.07 P = RASQSVSYQYLA P = GASSRATP = QQYGSVPR (SEQ ID NO: 252) (SEQ ID NO: 253) (SEQ ID NO: 254) L1a.08P = SGDSLGSYYVH P = DDNDRPS P = SAYDYSART (SEQ ID NO: 255)(SEQ ID NO: 256) (SEQ ID NO: 257) L1a.09 P = SGDNLGSKYVS P = DDDDRPS P =SSYDFLNIGL (SEQ ID NO: 258) (SEQ ID NO: 259) (SEQ ID NO: 260) L1a.10 P =SGDSLGKKSVH P = EDSERPS P = SSYTNSVD (SEQ ID NO: 261) (SEQ ID NO: 262)(SEQ ID NO: 263) L1a.11 P = SGDNLGKKYVG P = DDDNRPS P = QSYDDTSI(SEQ ID NO: 264) (SEQ ID NO: 265) (SEQ ID NO: 266) L1a.12 P =SGDSLGNKYVH P = DDSDRPS P = QTWDYVGY (SEQ ID NO: 267) (SEQ ID NO: 268)(SEQ ID NO: 269) L1a.13 P = TGTSSDVGGYNYVS P = DVSNRPS P = QSYDRYRLKN(SEQ ID NO: 270) (SEQ ID NO: 271) (SEQ ID NO: 272) *Determined by theKabat system (see supra). N = nucleotide sequence, P = polypeptidesequence.In certain embodiments, an antibody or antigen-binding fragmentcomprising the VL encoded by the polynucleotide specifically orpreferentially binds to Sp35.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region (VL)in which the CDR1, CDR2, and CDR3 regions have polypeptide sequenceswhich are identical to the CDR1, CDR2, and CDR3 groups shown in Table 5.In certain embodiments, an antibody or antigen-binding fragmentcomprising the VL encoded by the polynucleotide specifically orpreferentially binds to Sp35.

In a further aspect, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region (VL)in which the CDR1, CDR2, and CDR3 regions are encoded by nucleotidesequences which are identical to the nucleotide sequences which encodethe CDR1, CDR2, and CDR3 groups shown in Table 5. In certainembodiments, an antibody or antigen-binding fragment comprising the VLencoded by the polynucleotide specifically or preferentially binds toSp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VL encoded byone or more of the polynucleotides described above specifically orpreferentially binds to the same epitope as a monoclonal antibodyselected from the group consisting of 201′, 3A3, 3A6, 1A7, 1G7, 2B10,2C11, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9,3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (L100), 38-D01(Li02), 35-E04 (Li03), 36-C09 (Li04), 30-A11 (Li05), 34-F02 (Li06),29-E07 (Li07), 34-G04 (Li08), 36-A12 (Li09), 28-D02 (Li10), 30-B01(Li11), 34-B03 (Li12), Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495(L1a.2), 3563 (L1a.3), 3564 (L1a.4), 3565 (L1a.5), 3566 (L1a.6), 3567(L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570 (L1a.10), 3571 (L1a.11), 3582(L1a.12), and 1968 (L1a.13), or will competitively inhibit such amonoclonal antibody from binding to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VL encoded byone or more of the polynucleotides described above specifically orpreferentially binds to an Sp35 polypeptide or fragment thereof, or aSp35 variant polypeptide, with an affinity characterized by adissociation constant (K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M,5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸M, 10⁻⁸ M, 5×10⁻⁹M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M,10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a VH at least 80%, 85%, 90% or 95% identical to areference VH polypeptide sequence selected from the group consisting ofSEQ ID NOs: 158 to 172, 372, 376, 380, and 384 shown in Table 6. Incertain embodiments, an antibody or antigen-binding fragment comprisingthe VH encoded by the polynucleotide specifically or preferentiallybinds to Sp35.

In another aspect, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid sequence encoding a VH having a polypeptide sequenceselected from the group consisting of SEQ ID NOs: 158 to 172, 372, 376,380 and 384 shown in Table 6. In certain embodiments, an antibody orantigen-binding fragment comprising the VH encoded by the polynucleotidespecifically or preferentially binds to Sp35.

TABLE 6 VH Polypeptide Sequences SEQ ID VH Sequence NO: Li02EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYEMIWVRQAPGKGLEWVSSIGP 158SGGLTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCVRIDDSSGWAFDIWGQGTTVTVSSASTKGPSVFPLAP Li09EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYSMVWVRQAPGKGLEWVSYIS 159PSGGKTMYADSVKGRFTISRDNSKNTFYLQMNSLRAEDTAVYYCARDSRRRYYDFWSGYHNYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAP Li06EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYPMDWVRQAPGKGLEWVSSIY 160SSGGSTVYADSIKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGDSDAFDIWGQGTMVTVSSASTKGPSVFPLAP Li05EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYAMGWVRQAPGKGLEWVSSIV 161SSGGYTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGDHNAFDIWGQGTMVTVSSASTKGPSVFPLAP Li04EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYNMGWVRQAPGKGLEWVSVIY 162PSGGGTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSIADDAFDIWGQGTMVTVSSASTKGPSVFPLAP Li08EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYEMVWVRQAPGKGLEWVSSIRS 163SGGATKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKESPDDYFDYWGQGTLVTVSSASTKGPSVFPLAP Li11EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMYWVRQAPGKGLEWVSSIST 164SGGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDTSDNDYYYMDVWGKGTTVTVSSASTKGPSVFPLAP Li10EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYPMVWVRQAPGKGLEWVSWIG 165PSGGVTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPYSSGWWDFDLWGRGTLVTVSSASTKGPSVFPLAP Li01EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYQMTWVRQAPGKGLEWVSSIY 166PSGGNTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASGTTEAVFDYWGQGTLVTVSSASTKGPSVFPLAP Li07EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYFMGWVRQAPGKGLEWVSSIS 167PSGGFTSYADSVKGRFTISRDNSKNTLYLQMNSLAAEDTAVYYCARDRHAFDIWGQGTMVTVSSASTKGPSVFPLAP Li03EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYPMEWVRQAPGKGLEWVSGIY 168PSGGSTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGQWLGDFDYWGQGTLVTVSSASTKGPSVFPLAP Li12EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYNMFWVRQAPGKGLEWVSRISS 169SGGMTMYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREALRPYCSGGSCYSDYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAP 1A7QVQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGW 170INTDTGEPTYTEDFQGRFAFSLETSASTVYLQFNNLKNEDTATYFCAREGVHF DYWGQGTTVTVSS 2F3EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWLDWVRQSPEKGLEWVAEIR 171SKANNHATNYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYFCTPSFAYW GQGTTVTVSS 3P1DQVQLQQSGAELARPGASVKLSCRASGYTFTSSWTQWVKQRPGQGLEWIGAIY 172 102CPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARHNSYG 3 MDYWGQGTSVTVSS and3P1E 11.3B 7 Li13 EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYEMYWVRQAPGKGLEWVSRIV372 SSGGFTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATEGDNDA FDIWGQGTTVTVSSLi32 EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYMMQWVRQAPGKGLEWVSSIS 376PSGGNTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYGY WFDPWGQGTLVTVSS Li33EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWVRQAPGKGLEWVSWIGP 380SGGITKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAREGHNDWY FDLWGRGTLVTVSS Li34EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYEMYWVRQAPGKGLEWVSGIY 384SSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAAILDW YFDLWGRGTLVTVSS

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a VH at least 80%, 85%, 90% or 95% identical to areference VH polypeptide sequence selected from the group consisting ofSEQ ID NOs: 158-172, 372, 376, 380, and 384. In certain embodiments, anantibody or antigen-binding fragment comprising the VH encoded by thepolynucleotide specifically or preferentially binds to Sp35.

In another aspect, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid sequence encoding a VH of the invention, selected from thegroup consisting of SEQ ID NOs: 158-172, 372, 376, 380, and 384. Incertain embodiments, an antibody or antigen-binding fragment comprisingthe VH encoded by the polynucleotide specifically or preferentiallybinds to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VH encoded byone or more of the polynucleotides described above specifically orpreferentially binds to the same epitope as a monoclonal antibodyselected from the group consisting of, (201′) 3A3, 3A6, 1A7, 1G7, 2B10,2C11, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9,3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (L100), 38-D01(Li02), 35-E04 (Li03), 36-C09 (Li04), 30-All (Li05), 34-F02 (Li06),29-E07 (Li07), 34-G04 (Li08), 36-A12 (Li09), 28-D02 (Li10), 30-B01(Li11), 34-B03 (Li12), Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495(L1a.2), 3563 (L1a.3), 3564 (L1a.4), 3565 (L1a.5), 3566 (L1a.6), 3567(L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570 (L1a.10), 3571 (L1a.11), 3582(L1a.12), and 1968 (L1a.13), or will competitively inhibit such amonoclonal antibody from binding to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VH encoded byone or more of the polynucleotides described above specifically orpreferentially binds to an Sp35 polypeptide or fragment thereof, or aSp35 variant polypeptide, with an affinity characterized by adissociation constant (K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×⁻¹¹M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In additional embodiments, the present invention includes an isolatedpolynucleotide which encodes a heavy chain variable region (V_(H)),where the polynucleotide comprises a V_(H) nucleic acid sequenceselected from the group consisting of SEQ ID NOs 173 to 184, 370, 374,378 and 382, as shown in Table 7. In certain embodiments, an antibody orantigen-binding fragment comprising the VH encoded by the polynucleotidespecifically or preferentially binds to Sp35.

TABLE 7 VH Polynucleotide Sequences SEQ ID VH Sequence NO: Li02GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 173GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTACTTACGAGATGATTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCGGTCCTTCTGGTGGCCTTACTTGGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCGCCATGTATTACTGTGTACGGATTGATGATAGTAGTGGTTGGGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCT TCCCGCTAGCACCCLi09 GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 174GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTATGTACTCTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATATCTCTCCTTCTGGTGGCAAGACTATGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTTTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATTCGAGACGCCGGTATTACGATTTTTGGAGTGGTTATCACAACTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGCACCC Li06GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 175GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTGAGTACCCTATGGATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTATTCTTCTGGTGGCTCTACTGTTTATGCTGACTCCATTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCCAGAGAGGGTGACTCTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGC TAGCACCC Li05GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 176GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACGCTATGGGTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCGTTTCTTCTGGTGGCTATACTGATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCCAGAGAGGGTGACCATAATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGC TAGCACCC Li04GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 177GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACAATATGGGTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCGGTACTCATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGTTCTATAGCAGATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGC TAGCACCC Li08GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 178GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCATTACGAGATGGTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCCGTTCTTCTGGTGGCGCTACTAAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGAAAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAAAGAGTCGCCAGACGACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGC TAGCACCC Li11GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 179GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACGCTATGTATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTCTACTTCTGGTGGCTATACTGGTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATACCAGCGATAATGACTACTACTAAATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGCACCC Li10GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 180GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTACTTACCCTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTGGATCGGTCCTTCTGGTGGCGTTACTGCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGACCCTATAGCAGTGGCTGGTGGGACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCT TCCCGCTAGCACCCLi01 GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 181GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTAAGTACAAGATGACTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCAATACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGTGGGACTACAGAGGCAGTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGC TAGCACCC Li07GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 182GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTATGTACTTTATGGGTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTCTCCTTCTGGTGGCTTTACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGATCGGCATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGCAC CC Li03GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 183GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCAGTACCCTATGGAGTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGTATCTATCCTTCTGGTGGCTCTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGCGGGGCAGTGGCTGGGGGACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCC CGCTAGCACCC Li12GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 184GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCAGTACAATATGTTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGTATCTCTTCTTCTGGTGGCATGACTATGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGAAGCGTTACGGCCTTATTGTAGTGGTGGTAGCTGCTACTCCGACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGCACCC Li13GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTA 370CGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCATTACGAGATGTATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGTATCGTTTCTTCTGGTGGCTTTACTAAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCAACAGAGGGTGATAATGATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC Li32GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTA 374CGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTGCTTACATGATGCAGTGGGTTCCCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTCTCCTTCTGGTGGCAATACTAAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGAGATTATGGATACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTCACCGTCTCAAGC Li33GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTA 378CGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTATTTACCCTATGTTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTGGATCGGTCCTTCTGGTGGCATTACTAAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAQATGAACAGCTTAAGGGCTGAGGACACAGCCACATATTACTGTGCGAGAGAGGGGCATAACGACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC Li34GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTA 382CGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTAATTACGAGATGTATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGTATCTATTCTTCTGGTGGCATTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCTAGGGCAGCCATCCTCGACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCAAGC

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of aVH-encoding nucleic acid at least 80%, 85%, 90% or 95% identical to areference nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 173-184, 370, 374, 378 and 382 of Table 7. In certainembodiments, the polynucleotide encodes a VH polypeptide whichspecifically or preferentially binds to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VH encoded byone or more of the polynucleotides described above specifically orpreferentially binds to the same epitope as a monoclonal antibodyselected from the group consisting of, (201′) 3A3, 3A6, 1A7, 1G7, 2B10,2C11, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9,3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (L100), 38-D01(Li02), 35-E04 (Li03), 36-C09 (Li04), 30-A11 (Li05), 34-F02 (Li06),29-E07 (Li07), 34-G04 (Li08), 36-A12 (Li09), 28-D02 (Li10), 30-B01(Li11), 34-B03 (Li12), Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495(L1a.2), 3563 (L1a.3), 3564 (L1a.4), 3565 (L1a.5), 3566 (L1a.6), 3567(L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570 (L1a.10), 3571 (L1a.11), 3582(L1a.12), and 1968 (L1a.13), or will competitively inhibit such amonoclonal antibody from binding to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VH encoded byone or more of the polynucleotides described above specifically orpreferentially binds to an Sp35 polypeptide or fragment thereof, or aSp35 variant polypeptide, with an affinity characterized by adissociation constant (K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M,10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a VL at least 80%, 85%, 90% or 95% identical to areference VL polypeptide sequence selected from the group consisting ofSEQ ID NOs: 273 to 286, 373, 377, 381 and 385, shown in Table 8. Incertain embodiments, an antibody or antigen-binding fragment comprisingthe VL encoded by the polynucleotide specifically or preferentiallybinds to Sp35.

In another aspect, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid sequence encoding a VL having a polypeptide sequenceselected from the group consisting of SEQ ID NOs: 273 to 286, 373, 377,381 and 385, shown in Table 8. In certain embodiments, an antibody orantigen-binding fragment comprising the VL encoded by the polynucleotidespecifically or preferentially binds to Sp35.

TABLE 8 VL Polypeptide Sequences SEQ ID VL Sequence NO: Li02FYSHSAQYELTQPPSVSVSPGQTASITCSGDKLGDKFASWYQQKAGQSPVLV 273IFQDRKRLSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDTNTVVFG GGTKLTVLGQPKAAPLi09 FYSHSAQDIQMTQSPSSLSAFVGDRVAITCRASQSIDTYLNWYQQKPGKAPK 274LLIYAASKLEDGVPSRFSGSGTGTDFTLTIRSLQPEDFGTYYCQQSYSPPLTFG GGTKVEIKRTVAAPLi06 FYSHSAQDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPN 275LLIYAASSLRTGVPSRFRGSGSGTDFTLTISSLQPEDFATYYCLQDYSYPLTFG QGTKLEIKRTVAAPLi05 FYSHSAQSVLTQPPSVSVAPGQTARISCGGDNIGSKSVHWYQQRPGQAPVLV 276VYDDYDRPSGIPERFSGSNSGDTAILTITRVEVGDEADFYCQVRDSRTEERVF GGGTKVTVLGQPKAAPLi08 FYSHSAQDIQMTQSPSSLSASVGDRVTITCQASQDISYYLNWYQQKPGKAPK 277VLIYDVSNLQTGVPSRFSGSASATDFTLTISSLQPEDIATYYCQQSDNLPLTFG GGTKVEIKRTVAAPLi11 FYSHSAQDIQMTQSPSSVSAPIGDRVTITCRASQEIANYLAWYQQKPGKAPK 278LLIYDTYTLQTDVPPRFSGSGSGTDFTLTISSLQPEDTATYFCQQADIFPLSFG GGTKVEIKRTVAAPLi10 FYSHSAQDIQMTQSPSSMSASVGDTVTITCRASQGIGNWLAWYQQKPGKAP 279TLLIYAASSLESGVPSRFTGSGSSSGIDFTLTISDLHPEDLATYYCQQAQTFPLT FGGGTRVDLKRTVAAPLi01 FYSHSAQDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPK 280LLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQADRFPAVTF GGGTKVEIKRTVAAPLi07 FYSHSAQSELTQPPSVSVSPGQTAIITCSGDQLGDKHVAWYQQKPGQSPVLVI 281YLDIKRPAGISERFSGSNSGNTATLTIRGTQAMDEADYYCQAWDIKTVFGGG TKLTVLSQPKAAP Li03FYSHSAQDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL 282LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQ GTKVEIKRTVAAP 1A7QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSK 283LASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPFTFGSGTKLEI K 2F3DIQMTQSPASLSASVGETVTITCRASGNIYNYLAWFQQKQGKSPQLLVYNAK 284TLPDGVPSRFSGSGSGTQYFLKINSLQPEDFGSYYCQHFWAIPYTFGGGTKLE IKR 3P1DDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPK 285 10.2CLLIYWASTRESGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCQNDYSYPLFT 3 FGSGTKLEIR 3P1E1DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKSYLTWYQQKPGQPPK 286 1.3B7LLIYWASTRESGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCQNDYSYPLF TFGSGTKLEIR Li13DIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS 373NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPMYTFGQGTK LEIK Li32DIQMTQSPDSLSASVGDRVTITCQASQDISYYLNWYQQKPGMAPKLLIYDA 377FILEGGAPSRFSGSGSGTDFSFTISNLQPEDIATYFCQQSDQLPVTFGQGTKVE IR Li33DIQMTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS 381NRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYDKWPLTFGGGTKV ELK Li34DIQMTQSPSSLSASVGDRVTITCHASQDISNYLSWYQQKPGKAPKLLIYDAF 385NLETGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQHYDNLPFTFGPGTRVA IR

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a VL at least 80%, 85%, 90% or 95% identical to areference VL polypeptide sequence selected from the group consisting ofSEQ ID NOs: 273 to 286, 373, 377, 381 and 385. In certain embodiments,an antibody or antigen-binding fragment comprising the VL encoded by thepolynucleotide specifically or preferentially binds to Sp35.

In another aspect, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid sequence encoding a VL of the invention, selected from thegroup consisting of SEQ ID NOs: 273 to 286, 373, 377, 381 and 385. Incertain embodiments, an antibody or antigen-binding fragment comprisingthe VL encoded by the polynucleotide specifically or preferentiallybinds to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VL encoded byone or more of the polynucleotides described above specifically orpreferentially binds to the same epitope as a monoclonal antibodyselected from the group consisting of 201′, 3A3, 3A6, 1A7, 1G7, 2B10,2C11, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9,3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (L100), 38-D01(Li02), 35-E04 (Li03), 36-C09 (Li04), 30-All (Li05), 34-F02 (Li06),29-E07 (Li07), 34-G04 (Li08), 36-A12 (Li09), 28-D02 (Li10), 30-B01(Li11), 34-B03 (Li12), Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495(L1a.2), 3563 (L1a.3), 3564 (L1a.4), 3565 (L1a.5), 3566 (L1a.6), 3567(L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570 (L1a.10), 3571 (L1a.11), 3582(L1a.12), and 1968 (L1a.13), or will competitively inhibit such amonoclonal antibody from binding to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VL encoded byone or more of the polynucleotides described above specifically orpreferentially binds to an Sp35 polypeptide or fragment thereof, or aSp35 variant polypeptide, with an affinity characterized by adissociation constant (K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M,10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In additional embodiments, the present invention includes an isolatedpolynucleotide which encodes a light chain variable region (V_(L)),where the polynucleotide comprises a V_(L) nucleic acid sequenceselected from the group consisting of SEQ ID NOs 185 to 194, 371, 375,379 and 383, as shown in Table 9. In certain embodiments, an antibody orantigen-binding fragment comprising the VL encoded by the polynucleotidespecifically or preferentially binds to Sp35.

TABLE 9 VL Polynucleotide Sequences SEQ ID VL Sequence NO: Li02TTCTATTCTCACAGTGCACAGTACGAATTGACTCAGCCACCCTCAGTGTCCGTGTC 185CCCAGGACAGACAGCCAGCATCACCTGCTCTGGAGATAAATTGGGGGATAAATTTGCTTCCTGGTATCAGCAGAAGGCAGGCCAGTCCCCTGTGCTGGTCATCTTTCAAGATAGGAAGCGTCTCTCAGGGATCCCTGAGCGATTCTCTCGCTCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTATTACTGTCAGGCGTGGGACACCAACACTGTGGTCTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCC Li09TTCTATTCTCACAGTGCACAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTC 186TGCATTTGTGGGAGACAGAGTCGCCATCACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCTGCATCCAAGTTGGAAGACGGGGTCCCATCAAGATTCAGTGGCAGTGGAACTGGGACAGATTTCACTCTCACCATCAGAAGTCTGCAACCTGAAGATTTTGGAACTTACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCA Li06TTCTATTCTCACAGTGCACAAGACATCCAGATGACCCAGTCTCCTTCCACCCTGTC 187TGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATGCTGCATCCAGTTTACGAACTGGGGTCCCATCAAGATTCAGGGGCAGTGGATCTGGCACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACGTATTACTGTCTACAAGATTACAGTTACCCTCTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCA Li05TTCTATTCTCACAGTGCACAGAGCGTCTTGACTCAGCCACCCTCGGTGTCAGTGGC 188CCCAGGCCAGACGGCCAGGATTTCCTGTGGGGGAGACAACATTGGAAGTAAGAGTGTCCACTGGTACCACCAGAGGCCAGGCCAGGCCCCTQTCCTGGTCGTGTATGATGATTATGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGGACACGGCCATCCTGACCATCACCAGGGTCGAAGTCGGGGATGAGGCCGACTTTTATTGTCAGGTGAGGGACAGCCGTACTGAGGAACGGGTGTTCGGCGGAGGGACCAAGGTGACCGTCTTAGGTCAGCCCAAGGCTGCCCCC Li08TTCTATTCTCACAGTGCACAAGACATCCAGATGACCCAGTCTCCATCTTCCCTGTC 189TGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGTTACTATTTAAATTGGTATCAGCAGAAGCCAGGGAAAGCCCCTAAGGTCCTGATCTACGATGTATCCAATTTGCAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGCGTCTGCGACAGATTTTACTCTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCGACATATTACTGTCAACAGTCTGATAATCTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATTAAACGAACTGTGGCTGCACCA Li11TTCTATTCTCACAGTGCACAAGACATCCAGATGACCCAGTCTCCATCTTCTGTGTC 190TGCACCTATAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGAGATTGCCAACTACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATACATACACTTTGCAGACTGACGTCCCACCGAGGTTCAGCGGCAGTGGTTCGGGGACAGATTTCACTCTCACTATCAGCAGCCTGCAGCCTGAAGATACTGCAACTTACTTTTGTCAACAGGCTGACATTTTCCCGCTCTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCA Li10TTCTATTCTCACAGTGCACAAGACATCCAGATGACCCAGTCTCCATCTTCCATGTC 191TGCTTCTGTAGGGGACACAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTGGCAACTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCAACTCTCCTGATCTATGCTGCATCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCACCGGCAGCGGCAGTTCCTCTGGGATAGATTTCACTCTCACCATCAGCGACCTGCACCCTGAAGATTTGGCAACTTACTATTGTCAACAGGCTCAGACTTTCCCGCTCACCTTCGGCGGAGGGACCAGGGTGGACCTCAAGCGAACTGTGGCTGCACCA Li01TTCTATTCTCACAGTGCACAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTC 192TGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAQCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTGACAGGTTCCCTGCGGTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCA Li07TTCTATTCTCACAGTGCACAGAGCGAATTGACTCAGCCACCCTCAGTQTCCGTGTC 193CCCAGGACAGACAGCCATCATCACCTGCTCTGGAGATCAGTTGGGTGACAAACATGTGGCTTGGTATCAACAGAAGCCAGGCCAGTCCCCTGTGCTGGTCATCTATCTAGACATTAAGAGGCCCGCAGGGATTTCTGAGCGATTCTCTGGCTCCAACTCTGGAAATACAGCCACTCTGACCATCAGAGGGACCCAGGCTATGGATGAAGCTGACTATTACTGTCAGGCGTGGGACATCAAGACGGTCTTCGGCGGGGGGACCAAGCTGACCGTCCTGAGTCAGCCCAAGCCTGCCCCC Li03TTCTATTCTCACAGTGCACAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTC 194TGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCA Li13GACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAG 371CCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA Li32GACATCCAGATGACCCAGTCTCCAGACTCCCTGTCTGCATCTGTTGGAGACAGAG 375TCACCATCACTTGCCAGGCGAGTCAAGACATTAGCTACTATTTAAATTGGTATCAGCAGAAACCAGGGATGGCCCCTAAACTCCTCATCTACGATGCCTTCATTTTGGAAGGAGGGGCCCCATCACGGTTCAGTGGGAGCGGCTCTGGGACAGATTTTTCTTTCACCATCAGCAATCTACAGCCTGAGGATATTGCAACTTATTTCTGTCAACAGTCTGATCAACTGCCCGTGACCTTCGGCCAAGGGACCAAGGTGQAAATCAGA Li33GACATCCAGATGACCCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAG 379CCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAGGATTTTGCAGTTTATTACTGTCAGCAGTATGATAAGTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA Li34GACATCCAGATGACCCAGTCTCCATCCTCCCTCTCTGCATCTGTAGGAGACAGAG 383TCACCATCACTTGCCATGCGAGTCAGGACATTAGCAACTATTTAAGTTGGTATCAGCAGAAACCAGGTAAAGCCCCTAAACTCCTGATCTACGATGCTTTCAATTTGGAGACAGGAGTCCCATCGAGGTTCAGTGGAAGTGGATCTQGCACAGATTTTACATTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACATATTACTGTCAGCACTATCATAATCTCCCATTCACTTTCGGCCCTGGGACCAGAGTGGCGATCAGA

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a VL at least 80%, 85%, 90%, or 95% identical to aVL polynucleotide selected from the group consisting of SEQ ID NOs:185-194, 371, 375, 379 and 383 of Table 9. In certain embodiments, thepolynucleotide encodes a VL polypeptide which specifically orpreferentially binds to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VL encoded byone or more of the polynucleotides described above specifically orpreferentially binds to the same epitope as a monoclonal antibodyselected from the group consisting of 201′, 3A3, 3A6, 1A7, 1G7, 2B10,2C11, 2F3, 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9,3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8, 3P4C8.2G9, 30-C12 (L100), 38-D01(Li02), 35-E04 (Li03), 36-C09 (Li04), 30-A11 (Li05), 34-F02 (Li06),29-E07 (Li07), 34-G04 (Li08), 36-A12 (Li09), 28-D02 (Li10), 30-B01(Li11), 34-B03 (Li12), Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495(L1a.2), 3563 (L1a.3), 3564 (L1a.4), 3565 (L1a.5), 3566 (L1a.6), 3567(L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570 (L1a.10), 3571 (L1a.11), 3582(L1a.12), and 1968 (L1a.13), or will competitively inhibit such amonoclonal antibody from binding to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VL encoded byone or more of the polynucleotides described above specifically orpreferentially binds to an Sp35 polypeptide or fragment thereof, or aSp35 variant polypeptide, with an affinity characterized by adissociation constant (K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M,10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

Any of the polynucleotides described above may further includeadditional nucleic acids, encoding, e.g., a signal peptide to directsecretion of the encoded polypeptide, antibody constant regions asdescribed herein, or other heterologous polypeptides as describedherein.

Also, as described in more detail elsewhere herein, the presentinvention includes compositions comprising the polynucleotidescomprising one or more of the polynucleotides described above. In oneembodiment, the invention includes compositions comprising a firstpolynucleotide and second polynucleotide wherein said firstpolynucleotide encodes a VH polypeptide as described herein and whereinsaid second polynucleotide encodes a VL polypeptide as described herein.Specifically a composition which comprises, consists essentially of, orconsists of a VH polynucleotide, as show in Table 7, and a VLpolynucleotide, as shown in Table 9, wherein said VH polynucleotide andsaid VL polynucleotide are selected from the group consisting of:

-   -   i) SEQ ID NO:173 and SEQ ID NO:185;    -   ii) SEQ ID NO:174 and SEQ ID NO:186;    -   iii) SEQ ID NO:175 and SEQ ID NO:187;    -   iv) SEQ ID NO:176 and SEQ ID NO:188;    -   v) SEQ ID NO:178 and SEQ ID NO:189;    -   vi) SEQ ID NO:179 and SEQ ID NO:190;    -   vii) SEQ ID NO:180 and SEQ ID NO:191;    -   viii) SEQ ID NO:181 and SEQ ID NO:192;    -   ix) SEQ ID NO:182 and SEQ ID NO:193;    -   x) SEQ ID NO:183 and SEQ ID NO:194;    -   xi) SEQ ID NO:370 and SEQ ID NO:371;    -   xii) SEQ ID NO:374 and SEQ ID NO:375;    -   xiii) SEQ ID NO:378 and SEQ ID NO:379; and    -   xiv) SEQ ID NO:382 and SEQ ID NO:385.

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotideswhich encode fusion polynucleotides, Fab fragments, and otherderivatives, as described herein, are also contemplated by theinvention.

The polynucleotides may be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides (e.g., as described in Kutmeieret al., BioTechniques 17:242 (1994)), which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an Sp35 antibody, orantigen-binding fragment, variant, or derivative thereof may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the antibody may be chemically synthesized or obtainedfrom a suitable source (e.g., an antibody cDNA library, or a cDNAlibrary generated from, or nucleic acid, preferably poly A+RNA, isolatedfrom, any tissue or cells expressing the antibody or other Sp35antibody, such as hybridoma cells selected to express an antibody) byPCR amplification using synthetic primers hybridizable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence to identify, e.g., a cDNAclone from a cDNA library that encodes the antibody or other Sp35antibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe Sp35 antibody, or antigen-binding fragment, variant, or derivativethereof is determined, its nucleotide sequence may be manipulated usingmethods well known in the art for the manipulation of nucleotidesequences, e.g., recombinant DNA techniques, site directed mutagenesis,PCR, etc. (see, for example, the techniques described in Sambrook etal., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds.,Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998),which are both incorporated by reference herein in their entireties), togenerate antibodies having a different amino acid sequence, for exampleto create amino acid substitutions, deletions, and/or insertions.

A polynucleotide encoding an Sp35 antibody, or antigen-binding fragment,variant, or derivative thereof can be composed of any polyribonucleotideor polydeoxyribonucleotide, which may be unmodified RNA or DNA ormodified RNA or DNA. For example, a polynucleotide encoding Sp35antibody, or antigen-binding fragment, variant, or derivative thereofcan be composed of single- and double-stranded DNA, DNA that is amixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, a polynucleotideencoding an Sp35 antibody, or antigen-binding fragment, variant, orderivative thereof can be composed of triple-stranded regions comprisingRNA or DNA or both RNA and DNA. A polynucleotide encoding an Sp35antibody, or antigen-binding fragment, variant, or derivative thereofmay also contain one or more modified bases or DNA or RNA backbonesmodified for stability or for other reasons. “Modified” bases include,for example, tritylated bases and unusual bases such as inosine. Avariety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

V. Sp35 Antibody Polypeptides

The present invention is further directed to isolated polypeptides whichmake up Sp35 antibodies, antigen binding fragments, variants orderivatives thereof. Sp35 antibodies of the present invention comprisepolypeptides, e.g., amino acid sequences encoding Sp35-specific antigenbinding regions derived from immunoglobulin molecules. A polypeptide oramino acid sequence “derived from” a designated protein refers to theorigin of the polypeptide. In certain cases, the polypeptide or aminoacid sequence which is derived from a particular starting polypeptide oramino acid sequence has an amino acid sequence that is essentiallyidentical to that of the starting sequence, or a portion thereof,wherein the portion consists of at least 10-20 amino acids, at least20-30 amino acids, at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe starting sequence.

In one embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VH), where at least one ofCDRs of the heavy chain variable region or at least two of the CDRs ofthe heavy chain variable region are at least 80%, 85%, 90% or 95%identical to reference heavy chain CDR1, CDR2 or CDR3 amino acidsequences from monoclonal Sp35 antibodies disclosed herein.Alternatively, the CDR1, CDR2 and CDR3 regions of the VH are at least80%, 85%, 90% or 95% identical to reference heavy chain CDR1, CDR2 andCDR3 amino acid sequences from monoclonal Sp35 antibodies disclosedherein. Thus, according to this embodiment a heavy chain variable regionof the invention has CDR1, CDR2, and CDR3 polypeptide sequences relatedto the groups shown in Table 4, supra. In certain embodiments, anantibody or antigen-binding fragment comprising the VH polypeptidespecifically or preferentially binds to Sp35.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VH) in which the CDR1, CDR2,and CDR3 regions have polypeptide sequences which are identical to theCDR1, CDR2, and CDR3 groups shown in Table 4. In certain embodiments, anantibody or antigen-binding fragment comprising the VH polypeptidespecifically or preferentially binds to Sp35.

In a further embodiment, the present invention includes an isolatedpolypeptide comprising, consisting essentially of, or consisting of a VHpolypeptide at least 80%, 85%, 90% 95% or 100% identical to a referenceVH polypeptide sequence selected from the group consisting of SEQ IDNOs:158 to 172, 372, 376, 380 and 384 as shown in Table 6. In certainembodiments, an antibody or antigen-binding fragment comprising the VHpolypeptide specifically or preferentially binds to Sp35.

In another aspect, the present invention includes an isolatedpolypeptide comprising, consisting essentially of, or consisting of a VHpolypeptide selected from the group consisting of SEQ ID NOs: 158 to172, 372, 376, 380 and 384 as shown in Table 6. In certain embodiments,an antibody or antigen-binding fragment comprising the VH polypeptidespecifically or preferentially binds to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a one or more ofthe VH polypeptides described above specifically or preferentially bindsto the same epitope as a monoclonal antibody selected from the groupconsisting of 201′, 3A3, 3A6, 1A7, 1G7, 2B10, 2C11, 2F3, 3P1D10.2C3,3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2,3P4C5.1D8, 3P4C8.2G9, 30-C12 (L100), 38-D01 (Li02), 35-E04 (Li03),36-C09 (Li04), 30-A11 (Li05), 34-F02 (Li06), 29-E07 (Li07), 34-G04(Li08), 36-A12 (Li09), 28-D02 (Li10), 30-B01 (Li11), 34-B03 (Li12),Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495 (L1a.2), 3563 (L1a.3), 3564(L1a.4), 3565 (L1a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 (L1a.8), 3569(L1a.9), 3570 (L1a.10), 3571 (L1a.11), 3582 (L1a.12), and 1968 (L1a.13),or will competitively inhibit such a monoclonal antibody from binding toSp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of one or more ofthe VH polypeptides described above specifically or preferentially bindsto an Sp35 polypeptide or fragment thereof, or a Sp35 variantpolypeptide, with an affinity characterized by a dissociation constant(K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M,10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M,10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M,5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M,or 10⁻¹⁵ M.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (VL), where at least one ofthe CDRs of the light chain variable region or at least two of the CDRsof the light chain variable region are at least 80%, 85%, 90% or 95%identical to reference heavy chain CDR1, CDR2, or CDR3 amino acidsequences from monoclonal Sp35 antibodies disclosed herein.Alternatively, the CDR1, CDR2 and CDR3 regions of the VL are at least80%, 85%, 90% or 95% identical to reference light chain CDR1, CDR2, andCDR3 amino acid sequences from monoclonal Sp35 antibodies disclosedherein. Thus, according to this embodiment a light chain variable regionof the invention has CDR1, CDR2, and CDR3 polypeptide sequences relatedto the polypeptides shown in Table 5, supra. In certain embodiments, anantibody or antigen-binding fragment comprising the VL polypeptidespecifically or preferentially binds to Sp35.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (VL) in which the CDR1, CDR2,and CDR3 regions have polypeptide sequences which are identical to theCDR1, CDR2, and CDR3 groups shown in Table 5. In certain embodiments, anantibody or antigen-binding fragment comprising the VL polypeptidespecifically or preferentially binds to Sp35.

In a further embodiment, the present invention includes an isolatedpolypeptide comprising, consisting essentially of, or consisting of a VLpolypeptide at least 80%, 85%, 90% or 95% identical to a reference VLpolypeptide sequence selected from the group consisting of SEQ IDNOs:273 to 286, 373, 377, 381 and 385, shown in Table 8. In certainembodiments, an antibody or antigen-binding fragment comprising the VLpolypeptide specifically or preferentially binds to Sp35.

In another aspect, the present invention includes an isolatedpolypeptide comprising, consisting essentially of, or consisting of a VLpolypeptide selected from the group consisting of SEQ ID NOs: 273 to286, 373, 377, 381 and 385, shown in Table 8. In certain embodiments, anantibody or antigen-binding fragment comprising the VL polypeptidespecifically or preferentially binds to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, one or more of the VLpolypeptides described above specifically or preferentially binds to thesame epitope as a monoclonal antibody selected from the group consistingof 201′, 3A3, 3A6, 1A7, 1G7, 2B10, 2C11, 2F3, 3P1D10.2C3, 3P1E11.3B7,3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1D8,3P4C8.2G9, 30-C12 (L100), 38-D01 (Li02), 35-E04 (Li03), 36-C09 (Li04),30-A11 (Li05), 34-F02 (Li06), 29-E07 (Li07), 34-G04 (Li08), 36-A12(Li09), 28-D02 (Li10), 30-B01 (Li11), 34-B03 (Li12), Li13, Li32, Li33,Li34, 3383 (L1a.1), 3495 (L1a.2), 3563 (L1a.3), 3564 (L1a.4), 3565(L1a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570(L1a.10), 3571 (L1a.11), 3582 (L1a.12), and 1968 (L1a.13), or willcompetitively inhibit such a monoclonal antibody from binding to Sp35.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a one or more ofthe VL polypeptides described above specifically or preferentially bindsto an Sp35 polypeptide or fragment thereof, or a Sp35 variantpolypeptide, with an affinity characterized by a dissociation constant(K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M,10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M,10⁻⁸M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹²M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵M.

In other embodiments, an antibody or antigen-binding fragment thereofcomprises, consists essentially of or consists of a VH polypeptide, asshown in Table 6, and a VL polypeptide, as shown in Table 8, selectedfrom the group consisting of:

-   -   i) SEQ ID NO:170 and SEQ ID NO:283;    -   ii) SEQ ID NO:171 and SEQ ID NO:284;    -   iii) SEQ ID NO:172 and SEQ ID NO:285;    -   iv) SEQ ID NO:172 and SEQ ID NO:286;    -   v) SEQ ID NO:158 and SEQ ID NO:273;    -   vi) SEQ ID NO:159 and SEQ ID NO:274;    -   vii) SEQ ID NO:160 and SEQ ID NO:275;    -   viii) SEQ ID NO:161 and SEQ ID NO:276;    -   ix) SEQ ID NO:163 and SEQ ID NO:277;    -   x) SEQ ID NO:164 and SEQ ID NO:278;    -   xi) SEQ ID NO:165 and SEQ ID NO:279;    -   xii) SEQ ID NO:166 and SEQ ID NO:280;    -   xiii) SEQ ID NO:167 and SEQ ID NO:281;    -   xiv) SEQ ID NO:168 and SEQ ID NO:282;    -   xv) SEQ ID NO:372 and SEQ ID NO:373;    -   xvi) SEQ ID NO:376 and SEQ ID NO:377;    -   xvii) SEQ ID NO:380 and SEQ ID NO:381; and    -   xviii) SEQ ID NO:384 and SEQ ID NO:385.

Any of the polypeptides described above may further include additionalpolypeptides, e.g., a signal peptide to direct secretion of the encodedpolypeptide, antibody constant regions as described herein, or otherheterologous polypeptides as described herein. Additionally,polypeptides of the invention include polypeptide fragments as describedelsewhere. Additionally polypeptides of the invention include fusionpolypeptide, Fab fragments, and other derivatives, as described herein.

Also, as described in more detail elsewhere herein, the presentinvention includes compositions comprising the polypeptides describedabove.

It will also be understood by one of ordinary skill in the art that Sp35antibody polypeptides as disclosed herein may be modified such that theyvary in amino acid sequence from the naturally occurring bindingpolypeptide from which they were derived. For example, a polypeptide oramino acid sequence derived from a designated protein may be similar,e.g., have a certain percent identity to the starting sequence, e.g., itmay be 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the startingsequence.

Furthermore, nucleotide or amino acid substitutions, deletions, orinsertions leading to conservative substitutions or changes at“non-essential” amino acid regions may be made. For example, apolypeptide or amino acid sequence derived from a designated protein maybe identical to the starting sequence except for one or more individualamino acid substitutions, insertions, or deletions, e.g., one, two,three, four, five, six, seven, eight, nine, ten, fifteen, twenty or moreindividual amino acid substitutions, insertions, or deletions. Incertain embodiments, a polypeptide or amino acid sequence derived from adesignated protein has one to five, one to ten, one to fifteen, or oneto twenty individual amino acid substitutions, insertions, or deletionsrelative to the starting sequence.

Certain Sp35 antibody polypeptides of the present invention comprise,consist essentially of, or consist of an amino acid sequence derivedfrom a human amino acid sequence. However, certain Sp35 antibodypolypeptides comprise one or more contiguous amino acids derived fromanother mammalian species. For example, an Sp35 antibody of the presentinvention may include a primate heavy chain portion, hinge portion, orantigen binding region. In another example, one or more murine-derivedamino acids may be present in a non-murine antibody polypeptide, e.g.,in an antigen binding site of an Sp35 antibody. In certain therapeuticapplications, Sp35-specific antibodies, or antigen-binding fragments,variants, or analogs thereof are designed so as to not be immunogenic inthe animal to which the antibody is administered.

In certain embodiments, an Sp35 antibody polypeptide comprises an aminoacid sequence or one or more moieties not normally associated with anantibody. Exemplary modifications are described in more detail below.For example, a single-chain fv antibody fragment of the invention maycomprise a flexible linker sequence, or may be modified to add afunctional moiety (e.g., PEG, a drug, a toxin, or a label).

An Sp35 antibody polypeptide of the invention may comprise, consistessentially of, or consist of a fusion protein. Fusion proteins arechimeric molecules which comprise, for example, an immunoglobulinantigen-binding domain with at least one target binding site, and atleast one heterologous portion, i.e., a portion with which it is notnaturally linked in nature. The amino acid sequences may normally existin separate proteins that are brought together in the fusion polypeptideor they may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. Fusion proteins may be created,for example, by chemical synthesis, or by creating and translating apolynucleotide in which the peptide regions are encoded in the desiredrelationship.

The term “heterologous” as applied to a polynucleotide or a polypeptide,means that the polynucleotide or polypeptide is derived from a distinctentity from that of the rest of the entity to which it is beingcompared. For instance, as used herein, a “heterologous polypeptide” tobe fused to an Sp35 antibody, or an antigen-binding fragment, variant,or analog thereof is derived from a non-immunoglobulin polypeptide ofthe same species, or an immunoglobulin or non-immunoglobulin polypeptideof a different species.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in an immunoglobulin polypeptide is preferably replaced withanother amino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members.

Alternatively, in another embodiment, mutations may be introducedrandomly along all or part of the immunoglobulin coding sequence, suchas by saturation mutagenesis, and the resultant mutants can beincorporated into Sp35 antibodies for use in the diagnostic andtreatment methods disclosed herein and screened for their ability tobind to the desired antigen, e.g., Sp35.

VI. Fusion Proteins and Antibody Conjugates

As discussed in more detail elsewhere herein, Sp35 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention may further be recombinantly fused to a heterologouspolypeptide at the N- or C-terminus or chemically conjugated (includingcovalent and non-covalent conjugations) to polypeptides or othercompositions. For example, Sp35-specific Sp35 antibodies may berecombinantly fused or conjugated to molecules useful as labels indetection assays and effector molecules such as heterologouspolypeptides, drugs, radionuclides, or toxins. See, e.g., PCTpublications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387, which are incorporated herein by reference intheir entireties.

Sp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention include derivatives that are modified, i.e., bythe covalent attachment of any type of molecule to the antibody suchthat covalent attachment does not prevent the antibody binding Sp35. Forexample, but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including, but notlimited to specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. Additionally, the derivativemay contain one or more non-classical amino acids.

Sp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be composed of amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. Sp35-specific antibodies may be modified by naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in the Sp35-specific antibody,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini, or on moieties such as carbohydrates. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given Sp35-specificantibody. Also, a given Sp35-specific antibody may contain many types ofmodifications. Sp35-specific antibodies may be branched, for example, asa result of ubiquitination, and they may be cyclic, with or withoutbranching. Cyclic, branched, and branched cyclic Sp35-specificantibodies may result from posttranslation natural processes or may bemade by synthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, Proteins—Structure And Molecular Properties, T. E.Creighton, W. H. Freeman and Company, New York 2nd Ed., (1993);Posttranslational Covalent Modification Of Proteins, B. C. Johnson, Ed.,Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

The present invention also provides for fusion proteins comprising anSp35 antibody, or antigen-binding fragment, variant, or derivativethereof, and a heterologous polypeptide. The heterologous polypeptide towhich the antibody is fused may be useful for function or is useful totarget the Sp35 polypeptide expressing cells. In one embodiment, afusion protein of the invention comprises, consists essentially of, orconsists of, a polypeptide having the amino acid sequence of any one ormore of the V_(H) regions of an antibody of the invention or the aminoacid sequence of any one or more of the V_(L) regions of an antibody ofthe invention or fragments or variants thereof, and a heterologouspolypeptide sequence. In another embodiment, a fusion protein for use inthe diagnostic and treatment methods disclosed herein comprises,consists essentially of, or consists of a polypeptide having the aminoacid sequence of any one, two, three of the V_(H) CDRs of anSp35-specific antibody, or fragments, variants, or derivatives thereof,or the amino acid sequence of any one, two, three of the V_(L) CDRs ofan Sp35-specific antibody, or fragments, variants, or derivativesthereof, and a heterologous polypeptide sequence. In one embodiment, thefusion protein comprises a polypeptide having the amino acid sequence ofa V_(H) CDR3 of an Sp35-specific antibody of the present invention, orfragment, derivative, or variant thereof, and a heterologous polypeptidesequence, which fusion protein specifically binds to at least oneepitope of Sp35. In another embodiment, a fusion protein comprises apolypeptide having the amino acid sequence of at least one V_(H) regionof an Sp35-specific antibody of the invention and the amino acidsequence of at least one V_(L) region of an Sp35-specific antibody ofthe invention or fragments, derivatives or variants thereof, and aheterologous polypeptide sequence. Preferably, the V_(H) and V_(L)regions of the fusion protein correspond to a single source antibody (orscFv or Fab fragment) which specifically binds at least one epitope ofSp35. In yet another embodiment, a fusion protein for use in thediagnostic and treatment methods disclosed herein comprises apolypeptide having the amino acid sequence of any one, two, three ormore of the V_(H) CDRs of an Sp35-specific antibody and the amino acidsequence of any one, two, three or more of the V_(L) CDRs of anSp35-specific antibody, or fragments or variants thereof, and aheterologous polypeptide sequence. Preferably, two, three, four, five,six, or more of the V_(H)CDR(s) or V_(L)CDR(s) correspond to singlesource antibody (or scFv or Fab fragment) of the invention. Nucleic acidmolecules encoding these fusion proteins are also encompassed by theinvention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA84:2936-2940 (1987)); CD4 (Capon et al., Nature 337:525-531 (1989);Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990));L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991));CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley etal., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp.Med. 174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886(1991); and Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); and IgEreceptor a (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No.1448 (1991)).

In certain embodiments, Sp35 antibodies, antibody fragments, derivativesand variants thereof further comprise a targeting moiety. Targetingmoieties include a protein or a peptide which directs localization to acertain part of the body, for example, to the brain or compartmentstherein. In certain embodiments, Sp35 antibodies, antibody fragments,derivatives and variants thereof are attached or fused to a braintargeting moiety. The brain targeting moieties are attached covalently(e.g., direct, translational fusion, or by chemical linkage eitherdirectly or through a spacer molecule, which can be optionallycleavable) or non-covalently attached (e.g., through reversibleinteractions such as avidin, biotin, protein A, IgG, etc.). In otherembodiments, the Sp35 antibodies, antibody fragments, derivatives andvariants thereof are attached to one more brain targeting moieties. Inadditional embodiments, the brain targeting moiety is attached to aplurality of Sp35 antibodies, antibody fragments, derivatives andvariants thereof.

A brain targeting moiety associated with an Sp35 antibody, antibodyfragment, derivative or variant thereof enhances brain delivery of suchan Sp35 antibodies, antibody fragments, derivatives and variantsthereof. A number of polypeptides have been described which, when fusedto a protein or therapeutic agent, delivers the protein or therapeuticagent through the blood brain barrier (BBB). Non-limiting examplesinclude the single domain antibody FC5 (Abulrob et al. (2005) J.Neurochem. 95, 1201-1214); mAB 83-14, a monoclonal antibody to the humaninsulin receptor (Pardridge et al. (1995) Pharmacol. Res. 12, 807-816);the B2, B6 and B8 peptides binding to the human transferrin receptor(hTfR) (Xia et al. (2000) J. Virol. 74, 11359-11366); the OX26monoclonal antibody to the transferrin receptor (Pardridge et al. (1991)J. Pharmacol. Exp. Ther. 259, 66-70); and SEQ ID NOs: 1-18 of U.S. Pat.No. 6,306,365. The contents of the above references are incorporatedherein by reference in their entirety.

Enhanced brain delivery of an Sp35 antibody, antibody fragment,derivative or variant thereof is determined by a number of means wellestablished in the art. For example, administering to an animal aradioactively, enzymatically or fluorescently labeled Sp35 antibody,antibody fragment, derivative and variant thereof linked to a braintargeting moiety; determining brain localization; and comparinglocalization with an equivalent radioactively, enzymatically orfluorescently labeled Sp35 antibody, antibody fragment, derivative orvariant thereof that is not associated with a brain targeting moiety.Other means of determining enhanced targeting are described in the abovereferences.

As discussed elsewhere herein, Sp35 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention may befused to heterologous polypeptides to increase the in vivo half life ofthe polypeptides or for use in immunoassays using methods known in theart. For example, in one embodiment, PEG can be conjugated to the Sp35antibodies of the invention to increase their half-life in vivo. Leong,S. R., et al., Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev. 54:531(2002); or Weir et al., Biochem. Soc. Transactions 30:512 (2002).

Moreover, Sp35 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be fused to marker sequences,such as a peptide to facilitate their purification or detection. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

Fusion proteins can be prepared using methods that are well known in theart (see for example U.S. Pat. Nos. 5,116,964 and 5,225,538). Theprecise site at which the fusion is made may be selected empirically tooptimize the secretion or binding characteristics of the fusion protein.DNA encoding the fusion protein is then transfected into a host cell forexpression.

Sp35 antibodies or antigen-binding fragments, variants, or derivativesthereof of the present invention may be used in non-conjugated form ormay be conjugated to at least one of a variety of molecules, e.g., toimprove the therapeutic properties of the molecule, to facilitate targetdetection, or for imaging or therapy of the patient. Sp35 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be labeled or conjugated either before or afterpurification, when purification is performed.

In particular, Sp35 antibodies, or antigen-binding fragments, variants,or derivatives thereof of the invention may be conjugated to therapeuticagents, prodrugs, peptides, proteins, enzymes, viruses, lipids,biological response modifiers, pharmaceutical agents, or PEG.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared e.g.by reacting a binding polypeptide with an activated ester of biotin suchas the biotin N-hydroxysuccinimide ester. Similarly, conjugates with afluorescent marker may be prepared in the presence of a coupling agent,e.g. those listed herein, or by reaction with an isothiocyanate,preferably fluorescein-isothiocyanate. Conjugates of the Sp35antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention are prepared in an analogous manner.

The present invention further encompasses Sp35 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention conjugated to a diagnostic or therapeutic agent. The Sp35antibodies can be used diagnostically to, for example, monitor thedevelopment or progression of a neurological disease as part of aclinical testing procedure to, e.g., determine the efficacy of a giventreatment and/or prevention regimen. Detection can be facilitated bycoupling the Sp35 antibody, or antigen-binding fragment, variant, orderivative thereof to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, radioactivematerials, positron emitting metals using various positron emissiontomographies, and nonradioactive paramagnetic metal ions. See, forexample, U.S. Pat. No. 4,741,900 for metal ions which can be conjugatedto antibodies for use as diagnostics according to the present invention.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹Inor ⁹⁹Tc.

An Sp35 antibody, or antigen-binding fragment, variant, or derivativethereof also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedSp35 antibody is then determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of particularly useful chemiluminescent labeling compounds areluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt and oxalate ester.

One of the ways in which an Sp35 antibody, or antigen-binding fragment,variant, or derivative thereof can be detectably labeled is by linkingthe same to an enzyme and using the linked product in an enzymeimmunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay(ELISA)” Microbiological Associates Quarterly Publication, Walkersville,Md., Diagnostic Horizons 2:1-7 (1978)); Voller et al., J. Clin. Pathol.31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981);Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,(1980); Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin,Tokyo (1981). The enzyme, which is bound to the Sp35 antibody will reactwith an appropriate substrate, preferably a chromogenic substrate, insuch a manner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the Sp35 antibody,or antigen-binding fragment, variant, or derivative thereof, it ispossible to detect the antibody through the use of a radioimmunoassay(RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays,Seventh Training Course on Radioligand Assay Techniques, The EndocrineSociety, (March, 1986)), which is incorporated by reference herein). Theradioactive isotope can be detected by means including, but not limitedto, a gamma counter, a scintillation counter, or autoradiography.

An Sp35 antibody, or antigen-binding fragment, variant, or derivativethereof can also be detectably labeled using fluorescence emittingmetals such as 152Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

Techniques for conjugating various moieties to an Sp35 antibody, orantigen-binding fragment, variant, or derivative thereof are well known,see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting OfDrugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstromet al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview”, in Monoclonal Antibodies '84: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy”, in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16(1985), and Thorpe et al., “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

VII. Expression of Antibody Polypeptides

As is well known, RNA may be isolated from the original hybridoma cellsor from other transformed cells by standard techniques, such asguanidinium isothiocyanate extraction and precipitation followed bycentrifugation or chromatography. Where desirable, mRNA may be isolatedfrom total RNA by standard techniques such as chromatography on oligo dTcellulose. Suitable techniques are familiar in the art.

In one embodiment, cDNAs that encode the light and the heavy chains ofthe antibody may be made, either simultaneously or separately, usingreverse transcriptase and DNA polymerase in accordance with well knownmethods. PCR may be initiated by consensus constant region primers or bymore specific primers based on the published heavy and light chain DNAand amino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

Following manipulation of the isolated genetic material to provide Sp35antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention, the polynucleotides encoding the Sp35antibodies are typically inserted in an expression vector forintroduction into host cells that may be used to produce the desiredquantity of Sp35 antibody.

Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody which binds to atarget molecule described herein, e.g., Sp35, requires construction ofan expression vector containing a polynucleotide that encodes theantibody. Once a polynucleotide encoding an antibody molecule or a heavyor light chain of an antibody, or portion thereof (preferably containingthe heavy or light chain variable domain), of the invention has beenobtained, the vector for the production of the antibody molecule may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing a protein by expressing apolynucleotide containing an antibody encoding nucleotide sequence aredescribed herein. Methods which are well known to those skilled in theart can be used to construct expression vectors containing antibodycoding sequences and appropriate transcriptional and translationalcontrol signals. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, or a heavy or light chain thereof, or a heavy or light chainvariable domain, operably linked to a promoter. Such vectors may includethe nucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody may be cloned into such a vector for expression of the entireheavy or light chain.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain is advantageouslyplaced before the heavy chain to avoid an excess of toxic free heavychain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci.USA 77:2197 (1980)). The coding sequences for the heavy and light chainsmay comprise cDNA or genomic DNA.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells.

For the purposes of this invention, numerous expression vector systemsmay be employed. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells which have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals.

In particularly preferred embodiments the cloned variable region genesare inserted into an expression vector along with the heavy and lightchain constant region genes (preferably human) synthetic as discussedabove. In one embodiment, this is effected using a proprietaryexpression vector of Biogen IDEC, Inc., referred to as NEOSPLA (U.S.Pat. No. 6,159,730). This vector contains the cytomegaloviruspromoter/enhancer, the mouse beta globin major promoter, the SV40 originof replication, the bovine growth hormone polyadenylation sequence,neomycin phosphotransferase exon 1 and exon 2, the dihydrofolatereductase gene and leader sequence. This vector has been found to resultin very high level expression of antibodies upon incorporation ofvariable and constant region genes, transfection in CHO cells, followedby selection in G418 containing medium and methotrexate amplification.Of course, any expression vector which is capable of elicitingexpression in eukaryotic cells may be used in the present invention.Examples of suitable vectors include, but are not limited to plasmidspcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2,pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available fromInvitrogen, San Diego, Calif.), and plasmid pCI (available from Promega,Madison, Wis.). In general, screening large numbers of transformed cellsfor those which express suitably high levels if immunoglobulin heavy andlight chains is routine experimentation which can be carried out, forexample, by robotic systems. Vector systems are also taught in U.S. Pat.Nos. 5,736,137 and 5,658,570, each of which is incorporated by referencein its entirety herein. This system provides for high expression levels,e.g., >30 pg/cell/day. Other exemplary vector systems are disclosede.g., in U.S. Pat. No. 6,413,777.

In other preferred embodiments the Sp35 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention may beexpressed using polycistronic constructs such as those disclosed inUnited States Patent Application Publication No. 2003-0157641 A1, filedNov. 18, 2002 and incorporated herein in its entirety. In these novelexpression systems, multiple gene products of interest such as heavy andlight chains of antibodies may be produced from a single polycistronicconstruct. These systems advantageously use an internal ribosome entrysite (IRES) to provide relatively high levels of Sp35 antibodies, e.g.,binding polypeptides, e.g., Sp35-specific antibodies or immunospecificfragments thereof in eukaryotic host cells. Compatible IRES sequencesare disclosed in U.S. Pat. No. 6,193,980 which is also incorporatedherein. Those skilled in the art will appreciate that such expressionsystems may be used to effectively produce the full range of Sp35antibodies disclosed in the instant application.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the Sp35 antibody has been prepared, the expression vectormay be introduced into an appropriate host cell. Introduction of theplasmid into the host cell can be accomplished by various techniqueswell known to those of skill in the art. These include, but are notlimited to, transfection (including electrophoresis andelectroporation), protoplast fusion, calcium phosphate precipitation,cell fusion with enveloped DNA, microinjection, and infection withintact virus. See, Ridgway, A. A. G. “Mammalian Expression Vectors”Vectors, Rodriguez and Denhardt, Eds., Butterworths, Boston, Mass.,Chapter 24.2, pp. 470-472 (1988). Typically, plasmid introduction intothe host is via electroporation. The host cells harboring the expressionconstruct are grown under conditions appropriate to the production ofthe light chains and heavy chains, and assayed for heavy and/or lightchain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inpreferred embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains may be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

As used herein, “host cells” refers to cells which harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for the desired gene product to be expressed therein. Exemplaryhost cell lines include, but are not limited to, CHO (Chinese HamsterOvary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA(human cervical carcinoma), CVI (monkey kidney line), COS (a derivativeof CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293,WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast),HAK (hamster kidney line), SP2/O (mouse myeloma), P3×63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). CHO cells are particularly preferred. Host celllines are typically available from commercial services, the AmericanTissue Culture Collection or from published literature.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 1980) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al. (eds), Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.Biol. 150:1 (1981), which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Academic Press, New York, Vol. 3.(1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding Sp35 antibodies, or antigen-binding fragments, variants,or derivatives thereof of the invention can also be expressednon-mammalian cells such as bacteria or yeast or plant cells. Bacteriawhich readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologouspolypeptides must be isolated,purified and then assembled into functional molecules. Where tetravalentforms of antibodies are desired, the subunits will then self-assembleinto tetravalent antibodies (WO02/096948A2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris.

For expression in Saccharomyces, the plasmid YRp7, for example,(Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141(1979); Tschemper et al., Gene 10:157 (1980)) is commonly used. Thisplasmid already contains the TRP1 gene which provides a selection markerfor a mutant strain of yeast lacking the ability to grow in tryptophan,for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)). Thepresence of the trp1 lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.Alternatively, a preferred method for increasing the affinity ofantibodies of the invention is disclosed in US 2002 0123057 A1.

VIII. Treatment Methods Using Therapeutic Sp35 Antibodies

As described herein, Sp35 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can relieveNgR1-mediated inhibition of axonal extension that normally takes placein CNS neurons. This is beneficial in situations where axonal extensionor neurite sprouting is needed in the brain or spinal cord. Spinal cordinjury, including partial or complete crush or severance, exemplifies asituation in which axonal extension is needed, but is normally inhibitedthrough operation of the Nogo pathway. Examples of diseases or disordersin which axonal extension and/or neurite sprouting in the brain would bebeneficial include stroke, multiple sclerosis, and otherneurodegenerative diseases or disorders such as multiple sclerosis (MS),progressive multifocal leukoencephalopathy (PML), encephalomyelitis(EPL), central pontine myelolysis (CPM), adrenoleukodystrophy,Alexander's disease, Pelizaeus Merzbacher disease (PMZ), Globoid cellLeucodystrophy (Krabbe's disease) and Wallerian Degeneration, opticneuritis, transverse myelitis, amylotrophic lateral sclerosis (ALS),Huntington's disease, Alzheimer's disease, Parkinson's disease, spinalcord injury, traumatic brain injury, post radiation injury, neurologiccomplications of chemotherapy, stroke, neuropathy, acute ischemic opticneuropathy, vitamin E deficiency, isolated vitamin E deficiencysyndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome,metachromatic leukodystrophy, trigeminal neuralgia, Bell's palsy, spinalcord injury and all neurological diseases related to neuronal celldeath.

The inventors have further discovered that Sp35 is expressed inoligodendrocytes, and contributes to oligodendrocyte biology. Solublederivatives of Sp35, certain polynucleotides (e.g. RNAi), as well ascertain antibodies which specifically bind to Sp35, as described hereinact as antagonists to Sp35 function in oligodendrocytes, promotingproliferation, differentiation and survival of oligodendrocytes andpromoting myelination of neurons in vitro and in vivo. This isbeneficial in for diseases, disorders or conditions involvingdemyelination and dysmyelination. Examples of diseases or disorders inwhich oligodendrocyte proliferation, differentiation and survival,and/or myelination or remyelination would be beneficial include multiplesclerosis (MS), progressive multifocal leukoencephalopathy (PML),encephalomyelitis (EPL), central pontine myelolysis (CPM),adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease(PMZ), Globoid cell Leucodystrophy (Krabbe's disease), WallerianDegeneration, optic neuritis, transverse myelitis, amylotrophic lateralsclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson'sdisease, spinal cord injury, traumatic brain injury, post radiationinjury, neurologic complications of chemotherapy, stroke, acute ischemicoptic neuropathy, vitamin E deficiency, isolated vitamin E deficiencysyndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome,metachromatic leukodystrophy, trigeminal neuralgia, and Bell's palsy.

Accordingly, one embodiment of the present invention provides methodsfor treating spinal cord injury, diseases or disorders associated withinhibition of neuronal growth in the CNS, diseases or disordersassociated with inhibition of oligodendrocyte growth or differentiation,and diseases involving demyelination or dysmyelination of CNS neurons inan animal suffering from such injury or disease or predisposed tocontract such disease, the method comprising, consisting essentially of,or consisting of administering to the animal an effective amount of anSp35 antibody, or antigen-binding fragment, variant, or derivativethereof. Antibodies of the invention are described herein, and includethe monoclonal antibodies listed in Table 3A and 3B, antibodies whichspecifically bind to the same epitope as the monoclonal antibodieslisted in Table 3A and 3B, antibodies which competitively inhibitbinding of the monoclonal antibodies listed in Table 3A and 3B to Sp35,and antibodies comprising polypeptides derived from the monoclonalantibodies listed in Table 3A and 3B.

A therapeutic Sp35 antibody to be used in treatment methods disclosedherein can be prepared and used as a therapeutic agent which promotesCNS neurite outgrowth, neuronal survival, axon guidance and axonregeneration, which promotes oligodendrocyte survival, growth, and/ordifferentiation, and which promotes myelination or remyelination of CNSneurons. Characteristics of suitable therapeutic Sp35 antibodiesinclude: binding to Sp35 epitopes which result in blocking of Sp35activity, binding to Sp35 with sufficient affinity to elicit atherapeutic effect, and binding to Sp35 preferentially to normal bindingpartners, e.g., Nogo Receptor.

Therapeutic Sp35 antibodies may be monoclonal, chimeric or humanizedantibodies, or fragments of antibodies that bind specifically to Sp35.The antibodies may be monovalent, bivalent, polyvalent, or bifunctionalantibodies. Antibody fragments include without limitation Fab F(ab′)₂,and Fv fragments.

Therapeutic Sp35 antibodies, or antigen-binding fragments, variants orderivatives thereof according to the invention can be used in unlabeledor unconjugated form, or can be coupled or linked to drugs, labels orstabilization agents which may or may not exert additional therapeuticeffects.

A specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the particular Sp35antibody, or antigen-binding fragment, variant or derivative thereofused, the patient's age, body weight, general health, sex, and diet, andthe time of administration, rate of excretion, drug combination, and theseverity of the particular disease being treated. Judgment of suchfactors by medical caregivers is within the ordinary skill in the art.The amount will also depend on the individual patient to be treated, theroute of administration, the type of formulation, the characteristics ofthe compound used, the severity of the disease, and the desired effect.The amount used can be determined by pharmacological and pharmacokineticprinciples well known in the art.

In the methods of the invention the Sp35 antibodies, or antigen-bindingfragments, variants or derivatives thereof may be administered directlyto the nervous system, intracerebroventricularly, or intrathecally, e.g.into a chronic lesion of MS, as discussed in more detail below.

In various embodiments, an Sp35 antibody as described above is anantagonist of Sp35 activity. In certain embodiments, for example,binding of an antagonist Sp35 antibody to Sp35, as expressed on neurons,blocks myelin-associated neurite outgrowth inhibition or neuronal celldeath. In other embodiments, binding of the Sp35 antibody to Sp35, asexpressed on oligodendrocytes, blocks inhibition of oligodendrocytegrowth or differentiation, or blocks demyelination or dysmyelination ofCNS neurons.

In methods of the present invention, an Sp35 antibody, or anantigen-binding fragment, variant, or derivative thereof, in particularthe Sp35 antibodies described herein, can be administered directly as apreformed polypeptide, or indirectly through a nucleic acid vector, topermit beneficial axonal outgrowth, promote oligodendrocyteproliferation, differentiation, and survival, and/or promote myelinationor remyelination.

In certain embodiments, a subject may be treated with a nucleic acidmolecule encoding an Sp35 antibody, or antigen-binding fragment,variant, or analog thereof, e.g., in a vector. Doses for nucleic acidsencoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1μg to 10 mg, or 30-300 μg DNA per patient. Doses for infectious viralvectors vary from 10-100, or more, virions per dose.

In some embodiments of the present invention an Sp35 antibody, or anantigen-binding fragment, variant, or derivative thereof is administeredin a treatment method that includes: (1) transforming or transfecting animplantable host cell with a nucleic acid, e.g., a vector, thatexpresses an Sp35 antibody, or an antigen-binding fragment, variant, orderivative thereof; and (2) implanting the transformed host cell into amammal, at the site of a disease, disorder or injury. For example, thetransformed host cell can be implanted at the site of a spinal cordinjury or at a site of dysmyelination. In some embodiments of theinvention, the implantable host cell is removed from a mammal,temporarily cultured, transformed or transfected with an isolatednucleic acid encoding a an Sp35 antibody, and implanted back into thesame mammal from which it was removed. The cell can be, but is notrequired to be, removed from the same site at which it is implanted.Such embodiments, sometimes known as ex vivo gene therapy, can provide acontinuous supply of the Sp35 polypeptide, localized at the site of siteof action, for a limited period of time.

The methods for treating spinal cord injury, diseases or disordersassociated with inhibition of neuronal growth in the CNS, diseases ordisorders associated with inhibition of oligodendrocyte growth ordifferentiation, and diseases involving demyelination or dysmyelinationof CNS neurons comprising administration of an Sp35 antibody, orantigen-binding fragment, variant, or derivative thereof of theinvention are typically tested in vitro, and then in vivo in anacceptable animal model, for the desired therapeutic or prophylacticactivity, prior to use in humans. Suitable animal models, includingtransgenic animals, are will known to those of ordinary skill in theart. For example, in vitro assays to demonstrate the therapeutic utilityof Sp35 antibody described herein include the effect of an Sp35 antibodyon a cell line or a patient tissue sample. The effect of the Sp35antibody on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art, such as theassays disclosed elsewhere herein. In accordance with the invention, invitro assays which can be used to determine whether administration of aspecific Sp35 antibody is indicated, include in vitro cell cultureassays in which a patient tissue sample is grown in culture, and exposedto or otherwise administered a compound, and the effect of such compoundupon the tissue sample is observed.

Supplementary active compounds also can be incorporated into thecompositions of the invention. For example, a Sp35 antibody, orantigen-binding fragment, variant, or derivative thereof of theinvention may be coformulated with and/or coadministered with one ormore additional therapeutic agents.

The invention encompasses any suitable delivery method for a Sp35antibody, or antigen-binding fragment, variant, or derivative thereof ofthe invention to a selected target tissue, including bolus injection ofan aqueous solution or implantation of a controlled-release system. Useof a controlled-release implant reduces the need for repeat injections.

IX. Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering Sp35 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention to a subject in need thereof are well known to or are readilydetermined by those skilled in the art. The route of administration ofthe Sp35 antibody, or antigen-binding fragment, variant, or derivativethereof may be, for example, oral, parenteral, by inhalation or topical.The term parenteral as used herein includes, e.g., intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal orvaginal administration. While all these forms of administration areclearly contemplated as being within the scope of the invention, a formfor administration would be a solution for injection, in particular forintravenous or intraarterial injection or drip. Usually, a suitablepharmaceutical composition for injection may comprise a buffer (e.g.acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate),optionally a stabilizer agent (e.g. human albumin), etc. However, inother methods compatible with the teachings herein, Sp35 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be delivered directly to the site of the adverse cellularpopulation thereby increasing the exposure of the diseased tissue to thetherapeutic agent.

As previously discussed, Sp35 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention may be administered ina pharmaceutically effective amount for the in vivo treatment ofmammalian spinal cord injury, diseases or disorders associated withinhibition of neuronal growth in the CNS, diseases or disordersassociated with inhibition of oligodendrocyte growth or differentiation,and diseases involving demyelination or dysmyelination of CNS. In thisregard, it will be appreciated that the disclosed antibodies will beformulated so as to facilitate administration and promote stability ofthe active agent. Preferably, pharmaceutical compositions in accordancewith the present invention comprise a pharmaceutically acceptable,non-toxic, sterile carrier such as physiological saline, non-toxicbuffers, preservatives and the like. For the purposes of the instantapplication, a pharmaceutically effective amount of an Sp35 antibody, orantigen-binding fragment, variant, or derivative thereof, conjugated orunconjugated, shall be held to mean an amount sufficient to achieveeffective binding to a target and to achieve a benefit, e.g., toameliorate symptoms of a disease or disorder or to detect a substance ora cell.

The pharmaceutical compositions used in this invention comprisepharmaceutically acceptable carriers, including, e.g., ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Preparations for parenteral administration includes sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1M and preferably 0.05Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Suitableformulations for use in the therapeutic methods disclosed herein aredescribed in Remington's Pharmaceutical Sciences, Mack Publishing Co.,16th ed. (1980).

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., an Sp35 antibody, orantigen-binding fragment, variant, or derivative thereof, by itself orin combination with other active agents) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations may be packaged and sold in the form of a kit such as thosedescribed in co-pending U.S. Ser. No. 09/259,337 (US-2002-0102208 A1),which is incorporated herein by reference in its entirety. Such articlesof manufacture will preferably have labels or package inserts indicatingthat the associated compositions are useful for treating a subjectsuffering from, or predisposed to autoimmune or neoplastic disorders.

Parenteral formulations may be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionsmay be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis.

Certain pharmaceutical compositions used in this invention may be orallyadministered in an acceptable dosage form including, e.g., capsules,tablets, aqueous suspensions or solutions. Certain pharmaceuticalcompositions also may be administered by nasal aerosol or inhalation.Such compositions may be prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, absorption promoters toenhance bioavailability, and/or other conventional solubilizing ordispersing agents.

The amount of an Sp35 antibody, or fragment, variant, or derivativethereof that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host treated and theparticular mode of administration. The composition may be administeredas a single dose, multiple doses or over an established period of timein an infusion. Dosage regimens also may be adjusted to provide theoptimum desired response (e.g., a therapeutic or prophylactic response).

In keeping with the scope of the present disclosure, Sp35 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention may be administered to a human or other animal in accordancewith the aforementioned methods of treatment in an amount sufficient toproduce a therapeutic effect. The Sp35 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention can beadministered to such human or other animal in a conventional dosage formprepared by combining the antibody of the invention with a conventionalpharmaceutically acceptable carrier or diluent according to knowntechniques. It will be recognized by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.Those skilled in the art will further appreciate that a cocktailcomprising one or more species of Sp35 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention may proveto be particularly effective.

Effective doses of the compositions of the present invention, fortreatment of spinal cord injury, diseases or disorders associated withinhibition of neuronal growth in the CNS, diseases or disordersassociated with inhibition of oligodendrocyte growth or differentiation,and diseases involving demyelination or dysmyelination of CNS varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Usually, the patientis a human but non-human mammals including transgenic mammals can alsobe treated. Treatment dosages may be titrated using routine methodsknown to those of skill in the art to optimize safety and efficacy.

For treatment of spinal cord injury, diseases or disorders associatedwith inhibition of neuronal growth in the CNS, diseases or disordersassociated with inhibition of oligodendrocyte growth or differentiation,and diseases involving demyelination or dysmyelination of CNS with anSp35 antibody, or antigen-binding fragment, variant, or derivativethereof, the dosage can range, e.g., from about 0.0001 to 100 mg/kg, andmore usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg,0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. Forexample dosages can be 1 mg/kg body weight or 10 mg/kg body weight orwithin the range of 1-10 mg/kg, preferably at least 1 mg/kg. Dosesintermediate in the above ranges are also intended to be within thescope of the invention. Subjects can be administered such doses daily,on alternative days, weekly or according to any other scheduledetermined by empirical analysis. An exemplary treatment entailsadministration in multiple dosages over a prolonged period, for example,of at least six months. Additional exemplary treatment regimes entailadministration once per every two weeks or once a month or once every 3to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kgon consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly. Insome methods, two or more monoclonal antibodies with different bindingspecificities are administered simultaneously, in which case the dosageof each antibody administered falls within the ranges indicated.

Sp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be administered on multiple occasions.Intervals between single dosages can be daily, weekly, monthly oryearly. Intervals can also be irregular as indicated by measuring bloodlevels of target polypeptide or target molecule in the patient. In somemethods, dosage is adjusted to achieve a plasma polypeptideconcentration of 1-1000 μg/ml and in some methods 25-300 μg/ml.Alternatively, Sp35 antibodies, or antigen-binding fragments, variants,or derivatives thereof of the invention can be administered as asustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the antibody in the patient. The half-life of an Sp35antibody can also be prolonged via fusion to a stable polypeptide ormoeity, e.g., albumin or PEG. In general, humanized antibodies show thelongest half-life, followed by chimeric antibodies and nonhumanantibodies. In one embodiment, the Sp35 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention can beadministered in unconjugated form, In another embodiment, the Sp35antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be administered multiple times inconjugated form. In still another embodiment, Sp35 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be administered in unconjugated form, then in conjugatedform, or vice versa.

The compositions of the present invention may be administered by anysuitable method, e.g., parenterally, intraventricularly, orally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques. As describedpreviously, Sp35 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention act in the nervous system topromote survival, proliferation and differentiation of oligodendrocytesand myelination of neurons and neuronal survival, axon regeneration andaxon guidance. Accordingly, in the methods of the invention, the Sp35antibodies, or antigen-binding fragments, variants, or derivativesthereof are administered in such a way that they cross the blood-brainbarrier. This crossing can result from the physico-chemical propertiesinherent in the Sp35 antibody molecule itself, from other components ina pharmaceutical formulation, or from the use of a mechanical devicesuch as a needle, cannula or surgical instruments to breach theblood-brain barrier. Where the Sp35 antibody is a molecule that does notinherently cross the blood-brain barrier, e.g., a fusion to a moietythat facilitates the crossing, suitable routes of administration are,e.g., intrathecal or intracranial, e.g., directly into a chronic lesionof MS. Where the Sp35 antibody is a molecule that inherently crosses theblood-brain barrier, the route of administration may be by one or moreof the various routes described below. In some methods, antibodies areadministered as a sustained release composition or device, such as aMedipad™ device. Delivery across the blood brain barrier can be enhancedby a carrying molecule, such as anti-Fc receptor, transferrin,anti-insulin receptor or a toxin conjugate or penetration enhancer.

The Sp35 antibodies, or antigen-binding fragments, variants, orderivatives thereof used in the methods of the invention may be directlyinfused into the brain. Various implants for direct brain infusion ofcompounds are known and are effective in the delivery of therapeuticcompounds to human patients suffering from neurological disorders. Theseinclude chronic infusion into the brain using a pump, stereotacticallyimplanted, temporary interstitial catheters, permanent intracranialcatheter implants, and surgically implanted biodegradable implants. See,e.g., Gill et al., “Direct brain infusion of glial cell line-derivedneurotrophic factor in Parkinson disease,” Nature Med. 9: 589-95 (2003);Scharfen et al., “High Activity Iodine-125 Interstitial Implant ForGliomas,” Int. J. Radiation Oncology Biol. Phys. 24(4):583-91 (1992);Gaspar et al., “Permanent ¹²⁵I Implants for Recurrent MalignantGliomas,” Int. J. Radiation Oncology Biol. Phys. 43(5):977-82 (1999);chapter 66, pages 577-580, Bellezza et al., “Stereotactic InterstitialBrachytherapy,” in Gildenberg et al., Textbook of Stereotactic andFunctional Neurosurgery, McGraw-Hill (1998); and Brem et al., “TheSafety of Interstitial Chemotherapy with BCNU-Loaded Polymer Followed byRadiation Therapy in the Treatment of Newly Diagnosed Malignant Gliomas:Phase I Trial,” J. Neuro-Oncology 26:111-23 (1995).

The compositions may also comprise an Sp35 antibody dispersed in abiocompatible carrier material that functions as a suitable delivery orsupport system for the compounds. Suitable examples of sustained releasecarriers include semipermeable polymer matrices in the form of shapedarticles such as suppositories or capsules. Implantable or microcapsularsustained release matrices include polylactides (U.S. Pat. No.3,773,319; EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-56 (1985));poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate (Langer etal., J. Biomed. Mater. Res. 15:167-277 (1981); Langer, Chem. Tech.12:98-105 (1982)) or poly-D-(−)-3hydroxybutyric acid (EP 133,988).

In some embodiments of the invention, an Sp35 antibody, orantigen-binding fragment, variant, or derivative thereof of theinvention is administered to a patient by direct infusion into anappropriate region of the brain. See, e.g., Gill et al., supra.Alternative techniques are available and may be applied to administer anSp35 antibody according to the invention. For example, stereotacticplacement of a catheter or implant can be accomplished using theRiechert-Mundinger unit and the ZD (Zamorano-Dujovny) multipurposelocalizing unit. A contrast-enhanced computerized tomography (CT) scan,injecting 120 ml of omnipaque, 350 mg iodine/ml, with 2 mm slicethickness can allow three-dimensional multiplanar treatment planning(STP, Fischer, Freiburg, Germany). This equipment permits planning onthe basis of magnetic resonance imaging studies, merging the CT and MRItarget information for clear target confirmation.

The Leksell stereotactic system (Downs Surgical, Inc., Decatur, Ga.)modified for use with a GE CT scanner (General Electric Company,Milwaukee, Wis.) as well as the Brown-Roberts-Wells (BRW) stereotacticsystem (Radionics, Burlington, Mass.) can be used for this purpose.Thus, on the morning of the implant, the annular base ring of the BRWstereotactic frame can be attached to the patient's skull. Serial CTsections can be obtained at 3 mm intervals though the (target tissue)region with a graphite rod localizer frame clamped to the base plate. Acomputerized treatment planning program can be run on a VAX 11/780computer (Digital Equipment Corporation, Maynard, Mass.) using CTcoordinates of the graphite rod images to map between CT space and BRWspace.

Sp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can optionally be administered in combinationwith other agents that are effective in treating the disorder orcondition in need of treatment (e.g., prophylactic or therapeutic).

X. Diagnostics

The invention further provides a diagnostic method useful duringdiagnosis of neuronal disorders or injuries, which involves measuringthe expression level of Sp35 protein or transcript in tissue or othercells or body fluid from an individual and comparing the measuredexpression level with a standard Sp35 expression levels in normal tissueor body fluid, whereby an increase in the expression level compared tothe standard is indicative of a disorder.

Sp35-specific antibodies can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting proteinexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA), immunoprecipitation, or western blotting. Suitable assaysare described in more detail elsewhere herein.

By “assaying the expression level of Sp35 polypeptide” is intendedqualitatively or quantitatively measuring or estimating the level ofSp35 polypeptide in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level) or relatively (e.g.,by comparing to the cancer associated polypeptide level in a secondbiological sample). Preferably, Sp35 polypeptide expression level in thefirst biological sample is measured or estimated and compared to astandard Sp35 polypeptide level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” Sp35 polypeptide level is known, it can be used repeatedly asa standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source of cellspotentially expressing Sp35. Methods for obtaining tissue biopsies andbody fluids from mammals are well known in the art.

Sp35 antibodies for use in the diagnostic methods described aboveinclude any Sp35 antibody which specifically binds to an Sp35 geneproduct, as described elsewhere herein.

XI. Immunoassays

Sp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention may be assayed for immunospecific binding byany method known in the art. The immunoassays which can be used includebut are not limited to competitive and non-competitive assay systemsusing techniques such as western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork, Vol. 1 (1994), which is incorporated by reference herein in itsentirety). Exemplary immunoassays are described briefly below (but arenot intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4.degree. C., adding protein Aand/or protein G sepharose beads to the cell lysate, incubating forabout an hour or more at 4.degree. C., washing the beads in lysis bufferand resuspending the beads in SDS/sample buffer. The ability of theantibody of interest to immunoprecipitate a particular antigen can beassessed by, e.g., western blot analysis. One of skill in the art wouldbe knowledgeable as to the parameters that can be modified to increasethe binding of the antibody to an antigen and decrease the background(e.g., pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal., eds, Current Protocols in Molecular Biology, John Wiley & Sons,Inc., New York, Vol. 1 (1994) at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32p or 1251) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al., eds, Current Protocols in Molecular Biology, John Wiley & Sons,Inc., New York Vol. 1 (1994) at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley& Sons, Inc., New York, Vol. 1 (1994) at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest is conjugated to a labeled compound (e.g., ³H or¹²⁵I) in the presence of increasing amounts of an unlabeled secondantibody.

Sp35 antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention, additionally, be employed histologically, asin immunofluorescence, immunoelectron microscopy or non-immunologicalassays, for in situ detection of cancer antigen gene products orconserved variants or peptide fragments thereof. In situ detection maybe accomplished by removing a histological specimen from a patient, andapplying thereto a labeled Sp35 antibody, or antigen-binding fragment,variant, or derivative thereof, preferably applied by overlaying thelabeled antibody (or fragment) onto a biological sample. Through the useof such a procedure, it is possible to determine not only the presenceof Sp35 protein, or conserved variants or peptide fragments, but alsoits distribution in the examined tissue. Using the present invention,those of ordinary skill will readily perceive that any of a wide varietyof histological methods (such as staining procedures) can be modified inorder to achieve such in situ detection.

Immunoassays and non-immunoassays for Sp35 gene products or conservedvariants or peptide fragments thereof will typically comprise incubatinga sample, such as a biological fluid, a tissue extract, freshlyharvested cells, or lysates of cells which have been incubated in cellculture, in the presence of a detectably labeled antibody capable ofbinding to Sp35 or conserved variants or peptide fragments thereof, anddetecting the bound antibody by any of a number of techniques well-knownin the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled Sp35 antibody, orantigen-binding fragment, variant, or derivative thereof. The solidphase support may then be washed with the buffer a second time to removeunbound antibody. Optionally the antibody is subsequently labeled. Theamount of bound label on solid support may then be detected byconventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of Sp35 antibody, or antigen-bindingfragment, variant, or derivative thereof may be determined according towell known methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

There are a variety of methods available for measuring the affinity ofan antibody-antigen interaction, but relatively few for determining rateconstants. Most of the methods rely on either labeling antibody orantigen, which inevitably complicates routine measurements andintroduces uncertainties in the measured quantities.

Surface plasmon reasonance (SPR) as performed on BIAcore offers a numberof advantages over conventional methods of measuring the affinity ofantibody-antigen interactions: (i) no requirement to label eitherantibody or antigen; (ii) antibodies do not need to be purified inadvance, cell culture supernatant can be used directly; (iii) real-timemeasurements, allowing rapid semi-quantitative comparison of differentmonoclonal antibody interactions, are enabled and are sufficient formany evaluation purposes; (iv) biospecific surface can be regenerated sothat a series of different monoclonal antibodies can easily be comparedunder identical conditions; (v) analytical procedures are fullyautomated, and extensive series of measurements can be performed withoutuser intervention. BIAapplications Handbook, version AB (reprinted1998), BIACORE code No. BR-1001-86; BIAtechnology Handbook, version AB(reprinted 1998), BIACORE code No. BR-1001-84.

SPR based binding studies require that one member of a binding pair beimmobilized on a sensor surface. The binding partner immobilized isreferred to as the ligand. The binding partner in solution is referredto as the analyte. In some cases, the ligand is attached indirectly tothe surface through binding to another immobilized molecule, which isreferred as the capturing molecule. SPR response reflects a change inmass concentration at the detector surface as analytes bind ordissociate.

Based on SPR, real-time BIAcore measurements monitor interactionsdirectly as they happen. The technique is well suited to determinationof kinetic parameters. Comparative affinity ranking is extremely simpleto perform, and both kinetic and affinity constants can be derived fromthe sensorgram data.

When analyte is injected in a discrete pulse across a ligand surface,the resulting sensorgram can be divided into three essential phases: (i)Association of analyte with ligand during sample injection; (ii)Equilibrium or steady state during sample injection, where the rate ofanalyte binding is balanced by dissociation from the complex; (iii)Dissociation of analyte from the surface during buffer flow.

The association and dissociation phases provide information on thekinetics of analyte-ligand interaction (k_(a) and k_(d), the rates ofcomplex formation and dissociation, k_(d)/k_(a)=K_(D)). The equilibriumphase provides information on the affinity of the analyte-ligandinteraction (K_(D)).

BIAevaluation software provides comprehensive facilities for curvefitting using both numerical integration and global fitting algorithms.With suitable analysis of the data, separate rate and affinity constantsfor interaction can be obtained from simple BIAcore investigations. Therange of affinities measurable by this technique is very broad rangingfrom mM to pM.

Epitope specificity is an important characteristic of a monoclonalantibody. Epitope mapping with BIAcore, in contrast to conventionaltechniques using radioimmunoassay, ELISA or other surface adsorptionmethods, does not require labeling or purified antibodies, and allowsmulti-site specificity tests using a sequence of several monoclonalantibodies. Additionally, large numbers of analyses can be processedautomatically.

Pair-wise binding experiments test the ability of two MAbs to bindsimultaneously to the same antigen. MAbs directed against separateepitopes will bind independently, whereas MAbs directed againstidentical or closely related epitopes will interfere with each other'sbinding. These binding experiments with BIAcore are straightforward tocarry out.

For example, one can use a capture molecule to bind the first Mab,followed by addition of antigen and second MAb sequentially. Thesensorgrams will reveal: 1. how much of the antigen binds to first Mab,2. to what extent the second MAb binds to the surface-attached antigen,3. if the second MAb does not bind, whether reversing the order of thepair-wise test alters the results.

Peptide inhibition is another technique used for epitope mapping. Thismethod can complement pair-wise antibody binding studies, and can relatefunctional epitopes to structural features when the primary sequence ofthe antigen is known. Peptides or antigen fragments are tested forinhibition of binding of different MAbs to immobilized antigen. Peptideswhich interfere with binding of a given MAb are assumed to bestructurally related to the epitope defined by that MAb.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, D. N. Glover ed., Volumes 1 and 11 (1985); OligonucleotideSynthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds.(1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds.(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.,(1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors ForMammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer andWalker, eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986);Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

General principles of antibody engineering are set forth in AntibodyEngineering, 2nd edition, C. A. K. Borrebaeck, Ed., Oxford Univ. Press(1995). General principles of protein engineering are set forth inProtein Engineering, A Practical Approach, Rickwood, D., et al., Eds.,IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). General principlesof antibodies and antibody-hapten binding are set forth in: Nisonoff,A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, M A(1984); and Steward, M. W., Antibodies, Their Structure and Function,Chapman and Hall, New York, N.Y. (1984). Additionally, standard methodsin immunology known in the art and not specifically described aregenerally followed as in Current Protocols in Immunology, John Wiley &Sons, New York; Stites et al. (eds), Basic and Clinical-Immunology (8thed.), Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi(eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co.,New York (1980).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein, J., Immunology. The Science of Self-Nonself Discrimination, JohnWiley & Sons, New York (1982); Kennett, R., et al., eds., MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses, PlenumPress, New York (1980); Campbell, A., “Monoclonal Antibody Technology”in Burden, R., et al., eds., Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunnology4^(th) ed. Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A.Osborne, H. Freemand & Co. (2000); Roitt, I., Brostoff, J. and Male D.,Immunology 6^(th) ed. London: Mosby (2001); Abbas A., Abul, A. andLichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier HealthSciences Division (2005); Kontermann and Dubel, Antibody Engineering,Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Press (2001); Lewin, Genes VIII,Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR PrimerCold Spring Harbor Press (2003).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

EXAMPLES Example 1 Sp35 is Involved in Oligodendrocyte Biology

Oligodendrocytes mature through several developmental stages from A2B5progenitor cells (which express A2B5), differentiating intopre-myelinating oligodendrocytes (which express O1 and O4) and finallyinto mature myelinating oligodendrocytes (which express O1, O4 and MBP).Thus, by monitoring the presence and absence of the A2B5, O1, O4 and MBPmarkers it is possible to determine a given cell's developmental stageand to evaluate the role of Sp35-Fc in oligodendrocyte biology. For ageneral review of oligodendrocyte biology, see, e.g., Baumann andPham-Dinh, Physiol. Rev. 81: 871-927 (2001).

Monoclonal antibodies against O4, MBP and CNPase were from SternbergerMonoclonals; antibody to APC (clone CC-1; ref. 29) was from Calbiochem.Other antibodies were to βIII tubulin (Covance), Sp35 (Biogen Idec), Fyn(Santa Cruz Biotechnology) and phospho-Fyn (Biosource). Monoclonalantibodies against A2B5 are available from Chemicon.

Sp35 is Expressed in Oligodendrocytes

The expression of Sp35 in purified rat P13 CG neuron, P2oligodendrocyte, and P4 astrocyte cultures was analyzed by polymerasechain reaction after reverse transcription (RT-PCR). A kit from Ambion,Inc. was used to extract mRNA from the rat brain cells according to themanufacturer's instructions. Semi-quantitative RT-PCR was carried outusing forward primer 5′ AGAGACATGCGATTGGTGA 3′ (SEQ ID NO:344), andreverse primer 5′ AGAGATGTAGACGAGGTCATT 3′ (SEQ ID NO:345) showed highexpression in neurons, lower expression in oligodendrocytes, and noexpression in astrocytes.

The expression of Sp35 in oligodendrocytes was confirmed by in situhybridization in sections derived from adult rat optic nerve. Rat opticnerve sections were prepared and processed as described in Mi et al.,“Sp35 is a component of the Nogo-66 receptor/p75 signaling complex,”Nat. Neurosci. 7: 221-28 (2004) and probed with digoxigenin-labeled Sp35antisense or sense RNAs using the first 500 nucleotides of the Sp35coding sequence. The sections were stained according to themanufacturers' instructions using a Tyramide Signal Amplification kit(Amersham Biosciences) and a fluorescent anti-digoxigenin conjugatedantibody kit (Perkin Elmer). For combined in situ and immunofluorescenceanalyses, the sections were first probed with digoxigenin-labeled RNAsand then with antibodies, e.g. CC1 antibody (Calbiochem; a marker ofmature oligodendrocytes) or anti-Sp35 antibody. We observed thatoligodendrocytes that hybridized to an antisense Sp35 probe alsoco-stained with an antibody to CC1 (data not shown). No specificlabeling was observed using a sense Sp35 probe. Sp35 expression inoligodendrocytes also was confirmed by immunohistochemistry studies oftissue sections from the lateral ventricle region of P7 rat cortex. Amajority of cortical cells that labeled with CC1 antibody also labeledwith anti-Sp35 antibody. Data not shown. The specificity of theinteraction was confirmed by preadsorption of the anti-Sp35 antibodywith Sp35-Fc (see Example 2), which eliminated the signal.

Sp35-specific RNAi Knockdown of Sp35 Expression Promotes OligodendrocyteGrowth and Differentiation

Sp35-specific RNAi was used to ablate Sp35 expression in oligodendrocyteprecursor cells to examine how Sp35 contributes to oligodendrocytegrowth and differentiation. 50,000 A2B5 oligodendrocyte precursor cellswere infected with lentivirus carrying Sp35-specific RNAi sequence orcontrol RNAi prepared as follows.

Murine and rat Sp35 DNA sequences were compared to find homologousregions to use for candidate small-hairpin RNAs (shRNA). CH324, forlentivirus expression of Sp35 RNAi, was constructed by annealingoligonucleotides LV1-035 and LV1-036 and ligating to HpaI and XhoIdigested pLL3.7. The pLL3.7 vector, additional methodology and virusproduction were as described in Rubinson et al., Nat. Genet. 33, 401-06(2003). The Sp35 RNAi oligonucleotides were purchased from MWG and havethe following sequences: LV1-035 (sense oligo) 5′-TGA TCG TCA TCC TGCTAG ACT TCA AGA GAG TCT AGC AGG ATG ACG ATC TTT TTT C-3′ (SEQ ID NO:346)and LV1-036 (antisense oligo) 5′-TCG AGA AAA AAG ATC GTC ATC CTG CTA GACTCT CTT GAA GTC TAG CAG GAT GAC GAT CA-3′ (SEQ ID NO:347).

Control RNAi was designed with the same oligonucleotide sequences exceptfor the nucleotide changes indicated in lower-case letters: 5′-TGA TCcTCA TcC ttC Tat ACT TCA AGA GAG TgT AGC AGG ATG AcG ATC TTT TTT CTCGA-3′ (SEQ ID NO:348) and 5′-TCG AGA AAA AAG ATC GTC ATC CTG CTA GAC TCTCTT GAA GTa TAG aAG GAT GAC GAT CA-3′. (SEQ ID NO:349).

Prior to producing the lentivirus, DNA from pLL3.7 or candidate shRNA inpLL3.7 were cotransfected with murine Sp35-HA tagged plasmid at a ratioof 5 to 1 into CHO cells in a 6-well format. Knockdown was analyzed bywestern blot detection of Sp35-HA tag from transfected CHO cell lysatesas well as by northern blot of total RNA prepared from duplicate wells.The blot was probed with a fragment of Sp35 cDNA. Assays were performed48 hours post-transfection. As expected, there was a 10-fold reductionof Sp35 mRNA in CH324 RNAi-treated CHO cells relative to control-treatedcells. Data not shown. RNAi lentiviruses carrying green fluorescentprotein (GFP) were generated as described in Rubinson et al. In culturestreated with either control or Sp35 RNAi, approximately 80% of theoligodendrocytes were GFP positive. Total cell number was not altered bythe RNAi treatments. To quantify the effects of RNAi on differentiation,only GFP-expressing oligodendrocytes were counted.

Enriched populations of oligodendrocytes were grown from female LongEvans P2 rats as described by Conn, Meth. Neurosci. 2:1-4 (AcademicPress; 1990) with modifications as follows. Briefly, the forebrain wasdissected and placed in Hank's buffered salt solution (HBSS;Invitrogen). The tissue was cut into 1-mm fragments and was incubated at37° C. for 15 min in 0.01% trypsin and 10 μg/ml DNase. Dissociated cellswere plated on poly-L-lysine-coated T75 tissue culture flasks and weregrown at 37° C. for 10 d in DMEM medium with 20% fetal calf serum(Invitrogen). Oligodendrocyte precursors (A2B5⁺) were collected byshaking the flask overnight at 200 rpm at 37° C., resulting in a 95%pure population. Cultures were maintained in high-glucose Dulbecco'smodified Eagle's medium (DMEM) with FGF/PDGF (10 ng/ml; Peprotech) for 1week. Removal of FGF/PDGF allowed A2B5⁺ cells to differentiate into O4⁺premyelinating oligodendrocytes after 3-7 d, and to differentiate intoO4⁺ and MBP⁺ mature oligodendrocytes after 7-10 d. These differentiationstates are readily apparent from changes in morphology: A2B5⁺ cells arebipolar in shape, O4⁺ premyelinating oligodendrocytes have longer andmore branched processes and MBP⁺ mature oligodendrocytes contain myelinsheet structures between processes.

A2B5 oligodendrocyte precursor cells were infected with the lentiviruscontaining the CH324 RNAi. The resulting cells were cultured for 3 daysand the number of O4-positive (a marker for oligodendrocytedifferentiation) oligodendrocytes was counted. Endogenous Sp35expression was reduced by infection with Sp35 RNAi lentivirus and wasconfirmed by RT-PCR. Reduction of Sp35 resulted in more highlydifferentiated, mature oligodendrocytes as compared with controlinfected cells, as was evident by increases in the length of cellprocesses and by the presence of abundant myelin sheet structures (datanot shown). In cells that expressed Sp35 RNAi, there were three times asmany mature (O4-positive) oligodendrocytes as in control cultures. Thesedata indicate that Sp35 may negatively regulate oligodendrocytedifferentiation.

Dominant-Negative Sp35 Promotes Oligodendrocyte Growth andDifferentiation

Lentiviral vectors that express wild-type and a dominant-negative formof Sp35 were constructed. DNA sequence encoding mouse full length Sp35(FL-Sp35, amino acid residues 34-614 of SEQ ID NO:2) was amplified byPCR using primers 5′-GAG GAT CTC GAC GCG GCC GCA TGG AGA CAG ACA CAC TCCTG-3′ (SEQ ID NO:350) and 5′-GGG GCG GAA TTG GAT CCT CAC AGA TCC TCT TCTGAG ATG AG-3′ (SEQ ID NO:351) and inserted into the HRST-IRESeGFPlentiviral vector at the NotI and BamHI sites. Similarly, DNA sequenceencoding dominant negative Sp35 (DN-Sp35, amino acid residues 34-581 ofSEQ ID NO:2) was amplified by PCT using primers 5′-GAG GAT CTC GAC GCGGCC GCA TGG AGA CAG ACA CAC TCC TG-3′ (SEQ ID NO:352) and 5′-GAT ACG GATCCT CAG CCT TTG CCC CGG CTC CAT AGA AAC AGC-3′ (SEQ ID NO:353). TheFL-Sp35 and DN-Sp35 plasmids were transfected into 293 cells to producelentivirus as described by Rubinson et al., “A lentivirus-based systemto functionally silence genes in primary mammalian cells, stem cells andtransgenic mice by RNA interference,” Nat. Genet. 33: 401-06 (2003).Oligodendrocytes were infected with lentivirus at 2 MOI per cell andconfirmed expression of FL-Sp35 and DN-Sp35 by western blot.

DN-Sp35 promoted oligodendrocyte differentiation, producing an increasein the number of mature oligodendrocytes. In contrast, overexpression offull-length Sp35 (FL-Sp35) had the opposite effect and inhibiteddifferentiation, as was evident by a reduction in the number of matureoligodendrocytes as compared with the control (data not shown).

Example 2 Construction and Purification of Sp35-Fc Fusion Protein

A construct was made fusing the extra-cellular portion of human Sp35(residues 1-532) to the hinge and Fc region of human IgG1 to study thebiological function of Sp35. A partial coding sequence for human Sp35was obtained by PCR from clone 227.2 using the forward primer 5′-CAG CAGGTC GAC GCG GCC GCA TGC TGG CGG GGG GCG T-3′ (SEQ ID NO:354) and reverseprimer 5′-CAG CAG GTC GAC CTC GCC CGG CTG GTT GGC CAA CCA GCC GGG CGAGGT CGA CCT CGA GG-3′ (SEQ ID NO:355).

The blunt-end PCR product was subcloned into the SrfI site of the PCRSCRIPT AMP vector (Stratagene) to create PCR SCRIPT AMP-Sp35. A SalIfragment was isolated from PCR SCRIPT AMP-Sp35 and subcloned into thePCRCAMP Ig vector (derivative of Stratagene vector PCR SCRIPT AMP). Inthe PCRCAMP Ig vector, the hinge and Fc gamma sequence is subcloned as aSalI(5′) to NotI(3′) fragment. The SalI Sp35 fragment was subcloned intothe SalI site of the PCRCAMP Ig vector thereby fusing the Sp35 signalsequence and extracellular domain (codons 1-532) in-frame with sequencesencoding the hinge and Fc region of human Ig1. Correct isolates wereidentified, and a NotI fragment encompassing the Sp35 Fc fragment wassubcloned into the single NotI cloning site of the CHO expressionvector, PV90 (Biogen Idec). The resulting plasmid was confirmed by DNAsequencing and designated GT123.

Stable cell lines expressing the Sp35-Fc fusion protein were generatedby electroporation of CHO host cells DG44 with plasmid GT123.Transfected CHO cells were cultured in alpha minus MEM in the presenceof 10% dialyzed serum and 4 mM glutamine to select fornucleoside-independent growth. Fourteen days post-transfection, cellswere fed fresh media. To screen for cells expressing Sp35-Fc, CHO cellswere labeled with phycoerythrin (PE)-labeled goat anti-human IgG(Jackson Labs) and subjected to high speed flow cytometry sorting in aFACS Mo-Flo (Cytomation). The cells that expressed the highest levels ofSp35-Fc were selected. These cells were expanded in culture for 7 days,then re-labeled and re-sorted. Cells expressing the highest levels ofSp35-Fc were isolated as individual clones in 96-well plates. Theseclones were grown for two weeks and then fed fresh media one day priorto FACS analysis to check for expression levels. Clones that expressedthe highest levels of Sp35-Fc were expanded, and frozen cell banks wereestablished. The cell lines were adapted to grow in suspension culturein the serum-free media BCM16. The titer of Sp35-Fc produced by theseclones was determined by growing cell lines at 37° C. for 4-5 passages,then growing the cells to 50% maximal cell density and culturing themfor 10-15 days at 28° C. until the viable cell density dropped to 75%.At this time, the culture media were harvested, cleared of cells anddebris by centrifugation, and the culture supernatants titered forSp35-Fc levels by Western blot analysis using an anti-human Ig antibody(Jackson Lab) as the probe.

Sp35-Fc fusion protein was purified from the clarified culture medium asfollows: 9 ml of 1M HEPES pH 7.5 was added to 900 ml of conditionedmedium. The medium was batch loaded for 3 hr at 4° C. onto 3 ml ofProtein A Sepharose (Amersham Bioscience). The resin was collected in a1.5 cm (I.D.) column, and washed four times with 3 ml PBS, two timeswith 4 ml of PBS containing 800 mM NaCl, and then again with 3 ml ofPBS. The Sp35-Fc was eluted from the column with 25 mM NaH₂PO₄, pH 2.8and 100 mM NaCl in 1.5 ml fractions and neutralized by adding 75 μl of0.5 M NaH₂PO₄, pH 8.6. Peak protein-containing fractions were identifiedby absorbance at 280 nm, pooled, and subjected to further purificationon a 1 mL Protein A column. Prior to loading, NaCl was added to 600 mMand HEPES, pH 7.5 to 50 mM. The column was washed twice with 600 μl of10 mM HEPES pH 7.5 and 1 M NaCl, and then with 1 ml PBS. Sp35-Fc waseluted from the column with 25 mM NaH₂PO₄, pH 2.8 and 100 mM NaCl,collecting 0.5 mL fractions, and neutralized by adding 25 μl of 0.5 MNaH₂PO₄, pH 8.6. Peak protein-containing fractions were identified byabsorbance at 280 nm and pooled. By reducing SDS-PAGE, the Sp35-Fcprotein migrated as a single band (>95% pure) with an apparent mass of90 kDa. Under non-reducing conditions, the protein ran as a dimer withan approximate mass of 180 kDa. The purified Sp35-Fc protein wasaliquoted and stored at −70° C.

Example 3 Production of Sp35-Specific Monoclonal Antibodies

Anti-Sp35 Antibodies that specifically bind an Sp35 polypeptide of theinvention were made using the following methods and procedures.

A. Antibody Screening Assays

1. ELISA Assay

Sp35-Fc (0.5 μg in 50 μl of 0.1 M sodium bicarbonate buffer, pH 9.0) wasadded to each well of 96-well MaxiSorp™ plates (Nunc™). The plates werethen incubated at 37° C. for 1 hour or 4° C. for 16 hours. Non-specificbinding sites on the plates were blocked using 25 mM HEPES, pH 7.4containing 0.1% BSA, 0.1% ovalbumin, 0.1% (5% (w/v) nonfat dry milk in150 mM NACE) and 0.001% azide. Dilutions of serum or hybridomasupernatants (for example, serial three-fold dilutions) were addedacross each row of the plate, and incubated at 25° C. for 1 hour. Afterwashing three times with PBS, 50 μl of a 1:10,000 dilution ofhorseradish peroxidase-conjugated goat anti-mouse secondary antibody(Jackson ImmunoResearch Inc.) was added to each well and incubatedfurther for 1 hour. After three washings, color was developed by TMB(Pierce) and stopped with 2 M sulfuric acid. Color intensity wasmonitored in a spectrophotometer at 450 nm.

2. FACS Assay

COS-7 cells were labeled with 0.1 μM CellTracker™ Green CMFDA (MolecularProbes, Eugene, Oreg.) as described by the vendor. Equal volumes ofCellTracker™ labeled control cells were mixed with washed Sp35-COS-7cells (produced by transient transfection of Sp35 expression vector)before incubation with anti-Sp35 test sera or hybridoma supernatants.Fifty microliters of the cell mixture was dispensed into each well of a96-well V-bottom polystyrene plates (Costar® 3877, Corning, N.Y.) and100 μl of mouse serum, hybridoma supernatant, or a control anti-Sp35antibody was added. After incubation at 4° C. for 30 minutes, the cellswere washed and incubated with 50 μl of phycoerythrin-conjugatedaffinity pure F(ab′)₂ fragment goat anti-mouse IgG Fc gamma specificsecond antibody (1:200, Jackson ImmunoResearch Laboratory, West Grove,Pa.) in PBS. At the end of the incubation, the cells were washed twicewith PBS and suspended in 200 μl of PBS containing 1% fetal bovine serum(FBS), and subjected to FACS analyses. Alternately, Sp35-COS-7 cellswere mixed with mouse serum or hybridoma supernatant and then treatedwith R-phycoerythrin-conjugated goat anti-mouse secondary antibody anddirectly subjected to standard FACS analyses.

B. Hybridoma Production of Murine Monoclonal Anti-Sp35 Antibodies

Eight-week-old female RBF mice (Jackson Labs, Bar Harbor, Me.) wereimmunized intraperitoneally with emulsion containing 50 μg Sp35-Fc(amino acids 34 to 532 of SEQ ID NO:2 fused to the hinge and Fc regionof human IgG1), produced as described in Example 2 or were immunizedintraperitoneally with an emulsion containing 50 μg of human Sp35-Fc,and 50 μl complete Freund's adjuvant (Sigma® Chemical Co., St. Louis,Mo.) once every two weeks. Sera from the immunized mice were collectedbefore the first immunization and 1 week after the second and thirdimmunizations, and anti-Sp35 antibody titers were measured by FACS assayon Sp35-expressing COS-7 cells as described above. A booster final dosewas given after the third immunization and three days prior to whenhybridoma fusions were initiated.

Sera from mice immunized with the various Sp35 peptides were screened byELISA as described above. Mice that were positive for antibodies thatspecifically bound Sp35 expressing COS-7 cells were identified by flowcytometry (FACS) as described above, and were sacrificed. Splenocyteswere isolated from the mice and fused to the FL653 myeloma (anAPRT-derivative of a Ig-/HGPRT-Balb/c mouse myeloma, maintained in DMEMcontaining 10% FBS, 4500 mg/L glucose, 4 mM L-glutamine, and 20 mg/ml8-azaguanine) as described in Monoclonal Antibodies. Hybridomas: A NewDimension in Biological Analyses, ed. Kennett, R. H., McKearn, T. J. andBechtol, K. B. New York: Plenum Press (1982). Fused cells were platedinto 24- or 48-well plates (Corning Glass Works, Corning, N.Y.), and fedwith adenine, aminopterin and thymidine (AAT, available from Sigma®Chemical Co., St. Louis, Mo.) containing culture medium. AAT resistantcultures were screened by ELISA or flow cytometry as described above forbinding to either Sp35-COS-7 cells or to Sp35-Fc. Positive hybridomaswere further subcloned by limiting dilution.

Seventeen hybridoma cell lines producing monoclonal antibodies producedfrom mice immunized with Sp35-Fc were isolated. Properties of thehybridoma-derived monoclonal antibodies are shown in Tables 3A and 3B.

Polynucleotides encoding the variable domains (V_(H) and V_(L)) ofmonoclonal antibodies 1A7, 2F3, 3P1D10.2C3 and 3P1E11.3B7 were isolatedby PCR, cloned and were subjected to sequence analysis by the followingmethod. Total RNA was extracted from hybridoma cells using Qiagen®RNeasy® mini kit and cDNA was generated from the isolated RNA by RT PCR,using standard conditions. A cocktail of primers were used for theRT-PCR. A preferred set of primers included a primer with the 5′ of theprimer hybridizing to the signal sequence and the 3′ end of the primerhybridizing to the constant domain 3′ of the FR4/constant domainjunction. This allows for the amplification of an intact variable domainwith no ambiguities about the monoclonal antibody N-terminus and the V/Cjunction. One of skill in the art will recognize that primer sets needto be modified for amplifying different templates and for different PCRconditions. Occasionally, the presence of highly abundant nonproductivemessages (e.g. the CDR3-FR4 frameshifted nonproductive light chain fromthe fusion partner) or nonspecific productive messages can be producedand complicate the cloning of variable chains. One solution is to useN-terminal sequence data from the authentic purified antibody to designa degenerate primer to enable cloning. Alternatively, one can use“universal framework” primers, such as those described in Orlandi et al,PNAS 86:3833 (1989), which “fix” the N- and C-termini of the variabledomains (i.e. the N-terminus of FR1 and the C-terminus of FR4 areprimer-determined).

Additionally, sequence data, for designing more effective primers, canbe obtained from the bulk RT-PCR products which have been gel purifiedand then sequenced. The PCR product can also be subcloned using, forexample, the TOPO Cloning Kit (Invitrogen) then sequence. Sequence datais then obtained from multiple independent subclones or gel purifiedfragments to firmly establish the consensus sequence.

The sequence of the light chain of the P1E11.3B7 was determined by usinga cocktail of 5′ murine kappa light chain signal sequence primers: (i)5′ GGG GAT ATC CAC CAT GGA TTT TCA GGT GCA GAT TTT CAG 3′ (SEQ IDNO:356), (ii) 5′ GGG GAT ATC CAC CAT GRA GTC ACA KAC YCA GGT CTT YRT A3′ (SEQ ID NO:357), (iii) 5′ GGG GAT ATC CAC CAT GAA GTT GCC TGT TAG GCTGTT G 3′ (SEQ ID NO:358), and (iv) 5′ GGG GAT ATC CAC CAT GAG GKC CCCWGC TCA GYT YCT KGG A 3′ (SEQ ID NO:359), with a single 3′ murine kappaconstant domain primer: 5′ GCG TCT AGA ACT GGA TGG TGG GAG ATG GA 3′(SEQ ID NO:4), where K=G/T, R=A/G, W=A/T and Y=C/T. The resulting PCRproduct was subcloned and multiple independent subclones were sequenced.The deduced consensus sequence was consistent with the Edman degradationsequencing data. Sequencing indicated that the degenerate signalsequence 5′ primer 5′ GGG GAT ATC CAC CAT GRA GTC ACA KAC YCA GGT CTTYRT A 3′ (SEQ ID NO:357) was the one that had yielded the 3P1E11.3B7light chain variable domain during the amplification.

The 3P1E11.3B7 heavy chain sequence was determined using a cocktail ofmurine heavy chain signal sequence 5′ PCR primers: (i) 5′ GGG GAT ATCCAC CAT GGR ATG SAG CTG KGT MAT SCT CTT 3′, (SEQ ID NO:360) (ii) 5′ GGGGAT ATC CAC CAT GRA CTT CGG GYT GAG CTK GGT TTT 3′ (SEQ ID NO:361), and(iii) 5′ GGG GAT ATC CAC CAT GGC TGT CTT GGG GCT GCT CTT CT 3′ (SEQ IDNO:362), with a degenerate murine IgG CH1 constant domain 3′ primer 5′AGG TCT AGA AYC TCC ACA CAC AGG RRC CAG TGG ATA GAC 3′ (SEQ ID NO:363),where K=G/T, M=A/C, R=A/G, and Y=C/T. PCR using this cocktail ofprimers, with a variety of different cycling conditions, failed to yielda heavy chain variable domain sequence in which the deduced N-terminuswas consistent with that determined by Edman degradation sequence of thepurified 3P1E11.3B7 antibody. We therefore used heavy chain universalprimers: FR15′ AGG TSM ARC TGC AGS AGT CWG G 3′ (SEQ ID NO:364) andFR45′ TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CCA G 3′ (SEQ ID NO:365),where M=A/C, R=A/G, S=C/G, and W=A/T. This set yielded a murine heavychain variable domain whose deduced sequence was consistent with theempirical 3P1E11.3B7 data.

In order to verify that the heavy chain variable domain N- and C-terminiwere authentic and not primer-determined, another PCR reaction wasperformed with a degenerate signal sequence primer 5′ ATG GAR TGY AAYTGG ATH CTN CCN TTY A 3′ (SEQ ID NO:366) and the aforementioned constantdomain 3′ primer 5′ AGG TCT AGA AYC TCC ACA CAC AGG RRC CAG TGG ATA GAC3′ (SEQ ID NO:367), where H=A/C/T, N=A/C/G/T, R=A/G, and Y=C/T. Thedesign of the degenerate signal sequence primer was based upon signalsequences of the best hits derived from a TFASTA search of the Genbankrodent sequence database queried with the 3P1E11.3B7 consensus deducedFR1 sequence from the PCR reaction with the “universal primer” describedabove. This PCR yielded a product with a complete murine heavy chainvariable domain.

The complete 3P1E11.3B7 murine variable domains were used (with silentmutagenesis as necessary to introduce restriction sites) in conjunctionwith human IgG1 and kappa constant domain cDNAs to construct chimericheavy and light chain cDNAs, respectively. The full-lengthimmunoglobulin cDNAs were subcloned into an expression vector calledpNE001, a derivative of the commercial EBV mammalian cell episomalexpression vector pCEP4. The heavy and light chain expression vectors(called pXW372 and pXW363, respectively) were co-transfected into293-EBNA cells. Western blot analysis (probed with human IgG-specificreagents) of conditioned medium from transiently transfected cellsconfirmed the expression of chimeric 3P1E11.3B7-huIgG1, kappa mAb. Theresulting 3P1E11.3B7 VH and VL polypeptide sequences are shown in Tables6 and 8 and are SEQ ID NOs: 173 and 209, respectively. The heavy andlight chain sequences for the 1A7, 2F3, and 3P1D10.2C3 monoclonalantibodies were determined by similar methods.

C. Identification of Anti-Sp35 Monoclonal Antibodies by Phage Display

Anti-Sp35 monoclonal antibody Fab fragments were identified and isolatedfrom phage display libraries as described in Hoet et al., Nat. Biotech.23:344-348 (2005); Rauchenberger, et al., J. Biol. Chem. 278:194-205(2003); and Knappik, et al., J. Mol. Biol. 296:57-86 (2000), all ofwhich are incorporated herein by reference in their entireties.

The MorphoSys Fab-phage display library HuCAL® GOLD (“Phage DisplayLibrary-2” in Table 3B), which comprises humanized synthetic antibodyvariable regions was screened against recombinant human soluble Sp35-Fcprotein by standard ELISA AND IHC screening methods. See, e.g.,Ostendorp, R., Frisch, C. and Urban M, “Generation, engineering andproduction of human antibodies using HuCAL®.” Antibodies, Volume 2 NovelTechnologies and Therapeutic Use. New York: Kluwer Academic/Plenum 13-52(2004). Fab-phages that specifically bound to Sp35 were purified andcharacterized. Properties of these phage display-derived monoclonalantibody Fab fragments are shown in Table 3B as “phage displaylibrary-2-derived monoclonal Fab fragments.” Isolated Fab-phage 1968 wasselected for further analysis.

Example 4 Immunoprecipitation of Sp35 by Anti-Sp35 Monoclonal Antibodies

To perform the immunoprecipitation, COS-1 cells expressing Sp35, fusedto a hemaglutinin (HA) tag on the N-terminus, were produced bytransiently transfecting COS-1 cells with a DNA construct whichexpresses the full-length Sp35 protein with an HA tag. Cells wereharvested 48 hr after transfection and were lysed in 1 ml lysis buffer(50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 1% TritonX-100 and 10% glycerol) for 30 min at 4° C. After centrifugation at14,000×g for 15 min, the supernatants were incubated with ProteinA/G-Sepharose beads (Santa Cruz) at 4° C. for 1 hr, and then incubatedat 4° C. for 1 hr with either the 1A7 or the 2F3 anti-Sp35 murinemonoclonal antibodies. The beads were washed 3 times with lysis buffer,boiled in Laemmli sample buffer, subjected to 4-20% SDS-PAGE, andanalyzed by Western blotting using an antibody which recognizes the HAtag. As shown on the SDS-PAGE gel, monoclonal antibodies 1A7 and 2F3,immunoprecipitated human and murine Sp35 (FIG. 1). As shown in FIG. 1,monoclonal antibody 2F3 strongly immunoprecipitated both human andmurine Sp35, while monoclonal antibody 1A7, which stronglyimmunoprecipitated human Sp35, only recognized murine Sp35 proteinweakly. Similarly, monoclonal antibodies 1G7, 2B10, 2F3, 3P4C2.2D2,3P4C8.2G9, L100, Li03, Li05, Li06, Li07, Li08, Li11 and Li12immunoprecipitate human or mouse or human and mouse Sp35 (See Table 3B).Additionally, Li08 immunoprecipitates AP-Sp35 and monoclonal antibodies1B6.4 and 3E3.1 immunoprecipitate endogenous Sp35 (See Table 3B).

Example 5 Anti-Sp35 Antibody Binding Specifically to Sp35 Determined byELISA

In order to determine which regions of the Sp35 polypeptide were boundby the various hybridoma- and phage display-derived monoclonalantibodies produced in Example 2, an ELISA assay was performed using apanel of truncated Sp35 polypeptides, each fused to the hinge and Fcregions of IgG1 by the methods described in Example 1. The panelconsisted of the following Sp35 fragments: amino acids 34-425 of SEQ IDNO:2, amino acids 417-532 of SEQ ID NO:2, amino acids 417-493 of SEQ IDNO:2, and amino acids 34-532 of SEQ ID NO:2. Ovalbumin and BSA were usedas controls. As shown in Table 3B, hybridoma-derived mAbs 2F3, 2B10,3A3, 3P4c2.2d2, and 3P4c8.2 g9, and Fab-phage derived mAbs 3383, 3563,3564, 3565, 3568, 3569, 3570, and 3582 all specifically bound to the1-417 and 1-534 Sp35 fragments, suggesting that these antibodies bind toepitopes in the LRR region of Sp35. Hybridoma-derived Mabs 1A7,3P1B11F9, 3P1D10.2C3, 3P1E11.3B7, 3P2C63G10.2H7, 2P2C9.2G4, 3P4A61D9,and 394C51D8, and Fab-phage-derived Mabs 3495, 3566, 3567, and 1968specifically bound to the 34-532 Sp35 fragment and weakly bound to the417-532 Sp35, suggesting that these antibodies likely bind to epitopeswhich at least include a portion of Sp35 C-terminal to the LRR region.In similar experiments, these latter antibodies also specifically boundan Sp35 polypeptide consisting of amino acids 34-534 of human Sp35 andlow affinity to mouse and rat Sp35. The affinity of these latterantibodies for mouse and rat Sp35 was restored to the level seen usinghuman Sp35 when amino acid 419 of the mouse or rat Sp35 is changed fromhistidine (H) to arginine (R). Arginine is the amino acid at position419 in human Sp35. The K_(D) for monoclonal antibody 1A7 was determinedto be 10 nM (1×10⁻⁹M) for binding human Sp35 and 20 μM (2×10⁻⁵ M) forbinding murine Sp35. For Ap-Sp35 ELISA to detect the antibodies bound tothe 417 to 532 region, the ELISA was performed as follows: The Mabs werecoated onto ELISA plates, then incubated either with an Sp35-AP fusionprotein at 4° C. overnight followed by AP-linked anti-human (H+L)(1:5,000, Jackson ImmunoResearch) at RT for 1 hr, or with AP-fusionproteins at 4° C. overnight. AP substrate was then developed by 10 mg/ml4NPP in 0.1 M Glycin, 1 mM MgCl₂, 1 mM ZnCl₂, pH 10.5, and read at O.D.405.

Example 6 Anti-Sp35 Antibody Binding Specifically to Sp35 Determined byFACS

To further characterize the binding properties of hybridoma-derivedanti-Sp35 mAbs 1A7 and 2F3 produced as described in Example 3, bindingto both fixed and live COS-7 or 293 cells expressing mouse or human Sp35was compared. Sp35 transfected and non-transfected cells were fixed andsubject to FACS analysis (FACS: Cells transfected with human or mouseSp35 or vector control were dissociated from culture plates, washed with2% FBS/PBS, and incubated with primary antibody at 1 μg/ml on ice for 1hr. The cells were washed 3 times with 2% FBS/PBS, then incubated withPE labeled secondary antibody (1:100, JacksonImmunoResearch) on ice for30 min. After 2 washes with 2% FBS/PBS, cells were fixed in 2% PFA andsubjected to FACS analysis by PE.) FACS result showed that MAbs 1A7 and2F3 bound to COS-7 or 293 cells expressing Sp35, but not bind to controlcells with no Sp35 expression (FIG. 2).

Example 7 Neurite Outgrowth Assay

To test the ability of the hybridoma-derived and Fab-phage-derivedmonoclonal antibodies produced above to reverse the inhibitory effect ofCNS myelin inhibitors, e.g., OMgp, on neurons, Lab-Tek® culture slides(4 wells) were coated with 0.1 mg/ml poly-D-lysine (Sigma®). Ap-OMgp (1μg/spot) or PBS was spotted as 3 μl drops. Lab-Tek® slides were thenrinsed and coated with 10 μg/ml laminin (Gibco™). Dorsal root ganglions(DRG's) from P6-7 Sprague Dawley rat pups were dissociated with 1 mg/mlcollagenase type 1 (Worthington), triturated with fire-polished Pasteurpipettes pre-plated to enrich in neuronal cells and finally plated at10,000 cells/well on the pre-coated Lab-Tek® culture slides. Ten μg/mlof mAb 1A7 or 2F3 were added immediately after plating of the DRGs. Theculture medium was F12 (available from Gibco/Invitrogen) containing 5%heat inactivated donor horse serum, 5% heat inactivated fetal bovineserum and 50 ng/ml mouse nerve growth factor (mNGF) and incubated at 37°C. and 5% CO₂ for 6 hours. Following incubation, the slides were fixedin 4% paraformaldehyde/20% sucrose and stained with anti-βIII-tubulinTUJ1 antibody (Covance) after 16 hours.

As secondary antibody anti-mouse Alexa-Fluor® 594 (Molecular Probes)diluted 1:300 was added to the slides and incubated for 2 hours at roomtemperature. The slides were coverslipped with Gel/Mount™ (Biømeda™). 5×digital images were acquired with OpenLab™ software (Improvision, Inc.,Lexington, Mass.), and the images were analyzed for quantification ofneurite outgrowth using the OPENLAB™ software, all according tomanufacturer's specified parameters.

Both MAbs 1A7 and 2F3 protected DRG neurons from OMgp-mediatedinhibition of neurite outgrowth. (FIG. 3).

Example 8 Monoclonal Antibody 1A7 Promotes Functional Recovery in theRat Spinal Cord Injury Model

Spinal cord injury (“SCI”) was induced by dorsal over-hemi-section asfollows, modified from methods described previously (Li, S. et al. J.Neurosci. 24, 10511-10520 (2004)). Anesthetized female Long Evans rats(7 weeks old, Charles River) were given pre-operative analgesia(Buprenorphine/Buprenex, 0.05 mg/kg s.c.) and tranquillized (Midazolam,2.5 mg/kg i.p.) and a dorsal hemi-section was performed at thoracicvertebra 6/7 completely interrupting the main dorsomedial and thedorsolateral corticospinal tract (CST). The dorsal and dorso-lateralcomponents of the corticospinal tract (CST) were completely interruptedand the ventral portion of the CST left intact. The ventral tissuebridge remaining after hemi-section constituted approximately 20% of thecord in both treatment groups (data not shown).

Hindlimb function was quantified using the Basso-Beattie-Bresnahan (BBB)open field scoring method (Eby, M. T. et al., J. Biol. Chem. 275,15336-15342 (2000), incorporated herein by reference) and all animalssustained marked functional deficits after SCI, with almost completehindlimb paralysis the day after surgery. Immediately after CSTtransection, an intrathecal catheter was inserted into the subarachnoidspace at T7 and connected to a primed mini-osmotic pump (Alzet model2004, Alza Corp) inserted into the subcutaneous space. Mini-osmoticpumps delivered Human IgG isotype control protein (5 mg/ml) ormonoclonal antibody 1A7 (4.8 mg/ml) continuously at a rate of 0.25 μl/hover 5 weeks. Control (Human IgG-treated) animals recovered substantialfunction over the 5 week duration of the experiment, but plateaued at3-4 weeks, ultimately attaining a mean BBB score of 9±0.45 (FIG. 7). Incontrast, continuous intrathecal infusion of 1A7 for 5 weeks afterspinal cord transection resulted in significantly improved BBB scoresover the control animals by 5 weeks with a continued improvement infunction in the 2-5 week timeframe, reaching a mean BBB score of11.1±0.7 (FIG. 4). These results demonstrate that treatment withanti-Sp35 monoclonal antibody 1A7 promoted recovery of function afterspinal cord injury as demonstrated by an increase in BBB score, axonregeneration and less axon retraction observed by immunohistochemicalstaining of the axons.

Example 9 Anti-Sp35 Antibodies 1A7, 2F3, 3P1D10.2C3, 3P1E11.3B7,6P4F4.1D3, 6P4F4.1F9, 7P1D5.1G9, Li05, Li06, Li08, Li13, Li28, Li33, D05and D08 Promote Myelination In Vitro

The role of anti-Sp35 antibodies 1A7 and 2F3 in myelination wasinvestigated in vitro by treating co-cultures of dorsal root ganglion(DRG) neurons and oligodendrocytes with anti-Sp35 antibodies 1A7 and 2F3and testing for myelination by immunohistochemistry and Westernblotting. For these studies, it was necessary to first generate primarycultures of DRG neurons and of oligodendrocytes.

Female Long Evans rat E14-E17 embryonic dorsal root ganglia werecultured as described by Plant et al., J. Neurosci. 22:6083-91 (2002).Dissected DRGs were plated on poly-L-lysine-coated cover slips (100μg/ml) for 2 weeks. The cells were incubated in the presence offluorodeoxyuridine for days 2-6 and in NLA medium containing 1×B27, 100ng/ml NGF (Gibco) for days 8-11.

Female Long Evans post-natal day 2 (P2) rat oligodendrocytes werecultured as described by Conn, Meth. Neurosci. 2:1-4 (Academic Press;1990) with modifications as follows. Briefly, the forebrain wasextirpated from P2 rats and placed in cold HBSS medium (Gibco). Thetissue fragments were cut into 1 mm pieces and incubated at 37° C. for15 min in 0.01% trypsin and 10 μg/ml DNase. Dissociated cells wereplated on a poly-L-lysine coated T75 tissue culture flasks and grown inDMEM with 20% fetal bovine serum at 37° C. for 10 days. A2B5-positiveoligodendrocytes were collected by shaking the flasks overnight at 200rpm at 37° C. The A2B5 oligodendrocytes were cultured for 7 days in DMEM(Gibco) containing 25 mM D-glucose, 4 mM L-glutamine, 1 mM sodiumpyruvate, 50 μg/ml human apo-transferrin, 5 μg/ml bovine pancreaticinsulin, 30 nM sodium selenate, 10 nM hydrocortisone, 10 nM D-biotin, 1mg/ml BSA, 10 ng/ml FGF and PDGF (Peprotech). The cells were thenharvested by trypsinization. The cells then co-cultured with the DRGneurons in the presence or absence of 1, 3, 10, or 30 μg/ml of anti Sp35monoclonal antibodies 1A7 or 2F3, or a negative control antibody in NLAmedium containing 2% fetal bovine serum, 50 μg/ml ascorbic acid, 100ng/ml NGF (Gibco). An effective antibody dose to administer in such anassay has been determined to be in the range of 0.1 μg/ml to 10 μg/ml,depending upon the antibody. One of skill in the art would be able todetermine an effective dose using assays described herein.

The culture medium was changed and the various monoclonal antibodieswere replenished every three days. After 30 days at 37° C., theco-cultured cells were stained by immunohistochemical staining (“IHC”)for neurofilaments with anti-βIII-tubulin antibody to identify axons, oranti-MBP antibody to identify oligodendrocytes (FIG. 4A-E). Co-culturedcells were also lysed and subjected to Western blot analysis to quantifythe MBP (FIG. 4G). Based on IHC and Western blot analyses, co-culturedcells treated with anti-Sp35 antibodies 1A7 and 2F3 showed increasedsurvival of oligodendrocyte and neurons, increased numbers of bundledaxons and increased numbers of MBP positive cells (FIG. 4F, 10-fold moreMBP-positive cells when compared to control-antibody treatedco-cultures.

In a similar experiment, oligodendrocyte and DRG co-cultures wereincubated in the presence or absence of anti-Sp35 antibodies Li05 andLi06, or a negative control antibody. Co-cultured cells were lysed andsubjected to Western blot analysis to quantify the MBP (FIG. 8). Basedon Western blot analyses, co-cultured cells treated with anti-Sp35antibodies Li05 and Li06 showed increased numbers of MBP positive cells,similar to co-cultured cells treated with 3, 10 and 30 μg of Sp35-Fc(LINGO-1-Fc).

In similar experiments oligodendrocyte and DRG co-cultures wereincubated in the presence or absence of anti-Sp35 antibodies 3P1D10.2C3,3P1E11.3B7, 6P4F4.1D3, 6P4F4.1F9, 7P1D5.1G9, Li08, Li13, Li28, and Li33and also promoted myelination. Similarly, full-length antibodies D05 andD08 also promoted myelination.

These results indicated that treatment of DRG-oligodendrocyte cocultureswith anti-Sp35 antibodies 1A7, 2F3, 3P1D10.2C3, 3P1E11.3B7, 6P4F4.1D3,6P4F4.1F9, 7P1D5.1G9, Li05, Li06, Li08, Li13, Li28, Li33, D05 and D08promoted mature oligodendrocyte axon interactions and myelinationcompared to control-antibody treated co-cultures.

Example 10 Anti-Sp35 Antibody 1A7 Promotes Oligodendrocyte Survival andMyelination In Vivo

Adult wild-type C57BI/6 male mice were fed cuprizone (0.2% milled withground mouse chow by weight) for 6 weeks to induce demyelination withinthe corpus callosum according to the method described by Morell P etal., Mol Cell Neurosci. 12:220-7 (1998). Briefly, anti-Sp35 monoclonalantibody 1A7 was stereotactically injected into the demyelinating corpuscallosum at weeks 2, 2.5, and 3 weeks of cuprizone feeding, by themethod described below. Control mice were stereotactically injected atthe same intervals with sterilized media containing control antibody.After the 6 weeks of cuprizone feeding was completed, the mice werereturned to a normal diet for 2, 4 and 6 weeks (ground mouse chow only)to allow remyelination.

The 1A7 and control monoclonal antibodies were delivered as follows. Thecuprizone-treated mice were anesthetized with ketamine (80 mg/kg bodyweight) and xylazine (10 mg/kg body weight) and positioned in animmobilization apparatus designed for stereotactic surgery (David KopfInstruments). The scalp was opened and the sterile compounds injected (1μM in 1 ml of HBSS) unilaterally into the acutely demyelinated corpuscallosum of the wild-type recipient mice with a 10 μl Hamilton syringeusing stereotactic coordinates of 0.7 mm posterior and 0.3 mm lateral tobregma at a depth of 1.7 mm (Messier et al., Pharmacol. Biochem. Behav.63: 313-18 (1999)). Additional control recipient mice werestereotactically injected with HBSS containing no compounds. The openingin the skull was filled with Gelfoam, and the area was swabbed withpenicillin and streptomycin (Gibco) and the wound was sutured. Mice weresacrificed every week of the experiment after injection and their brainsremoved and processed for molecular, biochemical and histologicalanalysis.

The animals receiving anti-Sp35 antibody 1A7 treatment showed increasedmature oligodendrocyte survival (based on CC1 antibody staining, FIG.5A) and axon myelination by IHC using anti-MBP protein antibody or luxolfast blue (FIG. 5B). CC1 antibody-positive oligodendrocytes werequantitated at four weeks and 6 weeks (FIG. 5C). These results indicatedthat anti-Sp35 antibody 1A7 treatment promoted mature oligodendrocytesurvival and axon myelination compared to control-antibody treated mice.Similarly, animals receiving the 1A7 antibody in a lysolecithin model ofdemyelination also promoted axon myelination compared to control animals(data not shown).

Example 11 Anti-Sp35 Antibody 1A7 Promotes Retinal Ganglion Cell (RGC)Survival in the Optic Nerve Transection Model

Anti-Sp35 antibody 1A7 was tested in an optic nerve transection model,which investigates factors that affect neuronal function. Young adultfemale Sprague Dawley (SD) rats were used in this study. The right opticnerve of each animal was transected intraorbitally 1.5 mm from the opticdisc. A piece of gelfoam soaked with 6% Fluoro-Gold (FG) was applied tothe newly transected site right behind the optic disc to label thesurviving retinal ganglion cells (RGCs). The animals were divided intothree groups (n=6 in each group) which received either anti-Sp35antibody 1A7, control antibody, or just PBS, by intravitreal injection.The volume of each intravitreal injection was 4 μl while the dosage ofeach injection was 2 μg. The intravitreal injections were performedimmediately after the optic nerve transection.

All animals were allowed to survive for 1 week. Two days beforesacrificing the animals, the left optic nerve of each animal wastransected and 6% FG was administered as described above to label thesurviving RGCs, to serve as the internal control. Animals weresacrificed with an overdose of Nembutal and the retinas dissected in 4%paraformaldehyde. Four radial cuts were made to divide the retinas intofour quadrants (superior, inferior, nasal and temporal). The retinaswere then post-fixed in the same fixative for 1 hour before they wereflat-mounted with the mounting medium (Dako). The slides were examinedunder a fluorescence microscope using an ultra-violet filter (excitationwavelength=330-380 nm). Labeled RGCs were counted along the median lineof each quadrants starting from the optic disc to the peripheral borderof the retina at 500 μm intervals, under an eyepiece grid of 200×200μm². The percentage of surviving RGCs resulting from each treatment wasexpressed by comparing the number of surviving RGCs in the injured eyeswith their contra-lateral eyes. All data were expressed as mean±SEM.Statistical significance was evaluated by one way ANOVA, followed by aTukey-Kramer post hoc test. Differences were considered significant forp<0.05. Anti-Sp35 antibody 1A7 treated animals showed more neuronalsurvival (80%) when compared to control-antibody or PBS treated animals,which each only showed approximately 50% neuronal survival (FIG. 6).

Example 12 Testing Anti-Sp35 Antibodies for Remyelination in the OpticNerve Crush Model

The right optic nerve receives complete crush by #5 forceps for 10seconds around 1.5 mm behind the eyeball intraorbitally just beforeadministration of 2 μl of monoclonal antibody 1A7, 2F3, Li05 and Li06 in2 ml by intravitreal injection.

The animals receive a second intravitreal injection of the sametreatment one week after the surgery. Two weeks after the surgery, theanimals are perfused with EM fixatives, postfixed and processed forsemithin and ultrathin sections. The longitudinal optic nerve sectionsare stained and prepared for myelin observation. The myelination of theproximal and the distal parts of the crushed optic nerve are comparedamong different treatment groups. Sp35-Fc and 1A7, 2F3, Li05 and Li06treated animals, as well as appropriate controls, will be analyzed forremyelination in the distal part of the optic nerve compared to thecontrols.

Example 13 Testing Anti-Sp35 Antibodies for Axon Regeneration in theOptic Nerve Crush Model

The right optic nerve was crushed by #5 forceps for 10 seconds around1.5-2 mm behind the eyeball intraorbitally just before administration of2 μg of monoclonal antibody 1A7 in PBS via intravitreal injection. 4rats were tested with the 1A7 antibody and 8 rats were used as controlanimals. The animals received a second intravitreal injection of thesame treatment one week after the surgery. Three days prior to sacrificeof the test animals (day 11 of the experiment), 2 ml of CTB-FITC wasinjected intravitreally to label, anterograde, the regenerative opticnerve axons. On the 14th day post surgery, the animals were perfused andpostfixed. The crushed optic nerve was processed for frozen longitudinalsections. The CTB-FITC labeled axons, which cross the lesion site werecounted as regenerative fibers at various distances beyond the crushsite. When 1A7 was injected into the eye, regeneration of axons wasobserved up to 250 μm beyond the crush site. See FIG. 10.

Example 14 Anti-Sp35 Antibodies Promote Remyelination and Repair in theOptic Nerve Using the MOG Induced EAE Rat Model

For theses experiments, the Myelin Oligodendrocyte Glycoprotein (MOG)induced Experimental Autoimmune Encephalomyelitis (EAE) rat model wasused. This is the animal model for human multiple sclerosis. 50 μl of200 ng complete Freund's adjuvant (Chondrex Inc.) plus 50 μl of 50 μgMOG in saline was emulsified (1:1) and kept on ice before being injectedintradermally at the base of the tail for each animal. Female brownNorway rats, 8-10 weeks old, were used for all experiments. Generalobservation in the art indicates that the EAE model is induced around 15days after MOG injection. Rats are scored for clinical signs of EAE. Thesigns are scored as follows: grade 0.5, distal paresis of the tail;grade 1, complete tail paralysis; grade 1.5, paresis of the tail andmild hind leg paresis; grade 2.0, unilateral severe hind leg paresis;grade 2.5, bilateral severe hind limb paresis; grade 3.0, completebilateral hind limb paralysis; grade 3.5, complete bilateral hind limbparalysis and paresis of one front limb; grade complete paralysis(tetraplegia), moribund state, or death. The animals receive treatmentonce the EAE model is induced.

2 μg/μl of an anti-Sp35 antibody (1A7) was injected intravitreally atday 15 upon MOG-EAE induction. 2 μg/μl of the anti-Sp35 antibody, 1A7,was injected two additional times at day 22 and day 28. Upon terminationof the experiment, the animals were perfused with 4% PFA. The opticnerves were post fixed in 1% OsO₄, dehydrated and embedded in Epon.Semithin sections (1 μM) were cut and stained with Toluidine blue forevaluation of myelination. The optic nerves of treated animals werecompared to untreated animals for axon regeneration and remyelination inthe optic nerve. All procedures were performed following a protocolapproved by institutional animal care and use committee (IACUC).

Animals receiving treatment with the anti-Sp35 antibody 1A7 showedremyelination and repair of the optic nerve as compared to normal opticnerves or animals which were subjected to MOG-induced EAE, but receivedno treatment (FIG. 9). In FIG. 9C, the arrows point to myelinated axons.Animals receiving an antibody which recognizes domain III of Protein Gfrom Streptococcus (MOPC21), not specific for Sp35, showed no signs ofremylination or repair of the optic nerve as compared to normal opticnerves or the optic nerves of untreated animals (data not shown). TheSp35 antagonist antibody 1A7 promoted remyelination and repair of opticnerves in a rat MOG-induced EAE optic neuritis model (FIG. 9).

Example 15 Testing Anti-Sp35 Antibodies for Promotion of CNSRemyelination Using MOG Induced EAE Mouse Model

EAE is induced in the 129B6 mixed strain of mice by intradermalimmunization (day 0) with 100 μg MOG1-125 protein emulsified withcomplete Freund's adjuvant (CFA). The injected volume is 100 μl permouse and is distributed over 3 sites (pinnae, back and skin). Theemulsion is prepared on the basis of a 1:1 volume ratio and contains 1mg/ml MOG1-125 and 2 mg/ml M. tuberculosis (strain H37Ra, Chondrex).Pertussis toxin (200 ng/mouse) is administered intra-peritoneally at thetime of immunization and 2 days thereafter. Body weight and clinical EAEscores (0=no clinical signs; 1=limp tail; 2=hind limb weakness, impairedrighting reflex or waddled gait; 3=complete hind limb paralysis orabsent righting reflex; 4=complete hind limb paralysis with some degreeof fore limb involvement; 5=animal fully paralyzed; 6=moribund or dead)are recorded daily. All procedures are performed following a protocol,approved by our institutional animal care and use committee (IACUC). Theanimals receive the treatment with 1A7, 2F3, Li05 and Li06 monoclonalantibodies or control antibody at day 0 of the study. Blood samples aretaken at various times throughout the experiments by retro-orbitalbleeding technique. Plasma is separated from PBMC by centrifugation andcell phenotyping performed by FACS staining. Profiling of the humoralanti-MOG antibody response is performed by ELISA usingsubclass-/isotype-specific mAbs (Pharmingen). At the end of eachexperiment, brain, spinal cord, optic nerves and sciatic nerves areharvested following perfusion.

This same protocol is used to induce the EAE in Sp35 knockout mice andlitter mates. Sp35 knockout mice typically show lower EAE score (1.5),and no relapse compared to control (over a 45 day period), then wildtype litter mates (EAE score 3.5).

Sp35-Fc and 1A7, 2F3 treated animals will be analyzed for remyelinationcomparing to the control.

The His-tagged MOG₁₋₁₂₅ protein was expressed in Pichia pastoris using aDoxycycline inducible TetO-AOX1 promoter (M. Levesque, D. Krushinskieand K. Strauch, manuscript in preparation). The extracellular codingsequence (Gly1 through Gly125 of the mature protein after removal ofsignal sequence) of rat MOG was PCR amplified using the followingprimers: 5′ GGGGTATCTCTCGAGAAAAGAGAGCATCATCATCATCATCATATGGGACAGTTCAGAGTGATAGGG 3′ (SEQ ID NO:368), and 5′ TTCGCGGCCGCTATTAGCCAGGGTTGATCCAGTAGAAGGG3′ (SEQ ID NO:369).

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any compositions or methodswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

What is claimed is:
 1. An isolated monoclonal antibody or antigen-binding fragment thereof that can specifically bind to the same Sp35 epitope as a reference antibody comprising a VH region comprising the amino acids of SEQ ID NO:168, and a VL region comprising the amino acids of SEQ ID NO:282.
 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or fragment thereof can competitively inhibit the reference monoclonal antibody from specifically binding to Sp35.
 3. The antibody or fragment thereof of claim 1, which is an antagonist of Sp35-mediated myelination inhibition.
 4. The antibody or fragment thereof of claim 1, which is an antagonist of Sp35 mediated oligodendrocyte cell death.
 5. The antibody or fragment thereof of claim 1, which is an antagonist of Sp35 mediated oligodendrocyte differentiation inhibition.
 6. The antibody or fragment thereof of claim 1, further comprising a heterologous polypeptide fused thereto.
 7. The antibody or fragment thereof of claim 1, wherein said antibody is conjugated to an agent selected from the group consisting of a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.
 8. A composition comprising the antibody or fragment thereof of claim 1, and a carrier.
 9. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment comprises a VH region and a VL region, and wherein the VH region comprises the amino acids of SEQ ID NO:
 168. 10. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment comprises a VH region and a VL region, and wherein the VL region comprises the amino acids of SEQ ID NO:
 282. 11. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH region comprises the amino acids of SEQ ID NO: 168, and wherein the VL region comprises the amino acids of SEQ ID NO:
 282. 12. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment comprises a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 regions, and wherein the CDR1, CDR2, and CDR3 regions comprise the amino acids of SEQ ID NOs: 54, 56, and 58, respectively.
 13. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment comprises a VH region and a VL region, wherein the VL region comprises CDR1, CDR2, and CDR3 regions, and wherein the CDR1, CDR2, and CDR3 regions comprise the amino acids of SEQ ID NOs: 129, 131, and 133, respectively.
 14. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a VH region and a VL region, wherein the VH region comprises CDR1, CDR2, and CDR3 regions, and wherein the CDR1, CDR2, and CDR3 regions comprise the amino acids of SEQ ID NOs: 54, 56, and 58, respectively, and wherein the VL region comprises CDR1, CDR2, and CDR3 regions, and wherein the CDR1, CDR2, and CDR3 regions comprise the amino acids of SEQ ID NOs: 129, 131, and 133, respectively.
 15. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a brain targeting moiety.
 16. The antibody or antigen-binding fragment of claim 15, wherein the brain targeting moiety is selected from the group consisting of FC5, mAB 83-14, OX26, B2, B6, and B8 polypeptides, an anti-Fc receptor antibody, transferrin, an anti-transferrin receptor antibody, and an anti-insulin receptor antibody. 